Tlr8 transgenic animals

ABSTRACT

Provided herein are human Toll-like receptor 8 (TLR8)-expressing transgenic animals and methods of use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/434,805, filed Mar. 29, 2012, which claims the benefit of U.S.Provisional Patent Application No. 61/469,055, filed Mar. 29, 2011, bothof which are incorporated by reference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 377882005000SeqList.txt,date recorded: Mar. 29, 2012, size: 184 KB).

FIELD OF THE INVENTION

This application relates to human Toll-like receptor 8 (TLR8)-expressingtransgenic animals and methods of use thereof.

BACKGROUND OF THE INVENTION

Immunity can generally be classified as innate immunity or as adaptiveimmunity. Innate immune responses typically occur immediately uponinfection to provide an early barrier to infectious disease, whereasadaptive immune responses occur later with the generation ofantigen-specific effector cells and immunological memory. Innate immuneresponses do not generate lasting protective immunity, but appear toplay a role in the generation of later arising adaptive immuneresponses.

Toll-like receptors (TLRs) are essential for innate immune responses asthey recognize several different antigens and initiate immune responses(e.g., activation of dendritic cells and macrophages, and cytokineproduction). TLRs are type-I transmembrane proteins that recognize avariety of pathogen-associated molecular patterns (PAMPs) from bacteria,viruses and fungi. In this way PAMPs serve as a first-line of defenseagainst invading pathogens. Human TLRs can elicit overlapping yetdistinct biological responses due to differences in cellular expressionand activation of downstream signal transduction pathways (Akira et al.,Adv. Immunol. 78: 1-56, 2001).

TLRs are characterized by an ectodomain composed of leucine-rich repeatsand a cytoplasmic domain, known as a Toll/interleukin-1 receptor domain.The ectodomain is responsible for recognition of PAMPs, while thecytoplasmic domain is required for downstream signaling. TLRs usuallyundergo dimer formation and/or a conformation change to activatedownstream signal transduction pathways. Studies have shown that LRR8 isinvolved in DNA and RNA recognition, whereas LRR17 is involved innucleic acid binding (Smits et al., Oncologist, 13: 859-875, 2008).

The family of TLRs consists of ten members in human (TLR1-TLR10) andtwelve members in mice (TLR1-TLR9 and TLR11-TLR13). The TLRs that arelocated in the plasma membrane recognize bacterial membrane components,whereas the TLRs that detect nucleic acid-based ligands arepredominately located within endosomal compartments. The nucleicacid-sensing TLRs include TLR3, TLR7, TLR8, and TLR9. Uponligand-binding, TLRs initiate a signal transduction cascade leading toactivation of NFκB through the adapter protein myeloid differentiationprimary response gene 88 (MyD88) and recruitment of the IL-1 receptorassociated kinase (IRAK). Phosphorylation of IRAK in turn leads to therecruitment of TNF-receptor associated factor 6 (TRAF6), which resultsin the phosphorylation and degradation of the NF-κB inhibitor, I-κB,thereby releasing NF-κB. NF-κB enters the cell nucleus and initiatesgene transcription, leading to production of proflammatory cytokines,chemokines, and type I interferons (IFNs), as well as the upregulationof costimulatory molecules.

TLR8 belongs to the same subfamily as the TLR7 and TLR9 endosomalreceptors and is highly homologous to TLR7 (Liu et al., Mol Immunol.47:1083-90, 2010). The role of TLR8, and of its close homologue TLR7, isto detect the presence of “foreign” single-stranded RNA within a cell,as a means to respond to viral invasion (Heil et al., Science 303:1526,2004; and Diebold et al., Science 303:1529, 2004). While the TLR8 genein humans is closely related to TLR7, TLR8 has distinct, but overlappingspecificity for RNA and synthetic small molecules with a structurerelated to nucleic acids (Medzhitov et al., Immunol. Rev. 173:89-97,2000). Some ssRNA synthetic sequences containing repetitive A/U motifsare able to specifically activate TLR8 but not TLR7 (Gorden et al. JImmunol. 174:1259-68, 2005). Further, in humans, TLR8 is highlyexpressed in monocytes, macrophages, myeloid dendritic cells (mDC) andneutrophils, whereas TLR7 in blood cells is principally expressed inpDCs, B-cells, and neutrophils. Because of this difference in cellularexpression, triggering by RNA through TLR7 in blood leads to a responsedominated by Type I IFN production, whereas activation through TLR8induces multiple pro-inflammatory cytokines: TNF, IL-12, IL-6, IL-8 andIL-1 (Barrat et al., J Exp Med. 2202:1131-9, 2005; and Gorden et al., JImmunol. 174:1259-68, 2005).

Although TLR8 polymorphisms have been associated with some autoimmunediseases, the role of TLR8 and its specific ligands has not been clearlydefined. One key limitation in elucidating TLR8 biology is the lack ofan animal model. For example, mouse TLR8 has a very differentspecificity than human TLR8. Mouse TLR8 lacks the ability to respond tossRNA ligands, RNA viruses or small molecules; all of which have beenshown to activate human TLR8 (Heil et al. Science 303:1526-9, 2004; Jurket al. Nat Immunol 3:499, 2002; Hemmi et al. Nat. Immunol 3:196-200,2002; and Lund et al. PNAS 101:5598-603, 2004). Further, by comparingthe amino acid sequence, TLR8 of mice and rats lack a five amino-acidsequence required for ligand recognition in man (Liu et al. Mol Immunol.47:1083-90, 2010). The lack of useful animal models and the verydifferent ligand specificities of human TLR8 and its rodent orthologshave proven to be major limitations to the study of TLR8 biology.

SUMMARY OF THE INVENTION

Provided herein are transgenic animals comprising a nucleotide sequenceencoding human Toll-like receptor 8 (TLR8), wherein the human TLR8 isexpressed in the transgenic animal. In some embodiments, the transgenicanimal is a nonhuman mammal. In some embodiments, the transgenic animalis a small mammal selected from the group consisting of a mouse,hamster, rat, guineas pig, and rabbit. In some embodiments, thetransgenic animal is a mouse. In some embodiments, the transgenic animalis a chimeric transgenic animal. In some embodiments, both germ cellsand somatic cells of the transgenic animal comprise the nucleotidesequence encoding human TLR8. In some embodiments, the nucleotidesequence encoding human TLR8 is stably integrated into the genome of thetransgenic animal. In some embodiments, the nucleotide sequence encodinghuman TLR8 is present at a copy number of from 1 to 15 (e.g., from 1 to5, from 6 to 10, from 11 to 15, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15). In some embodiments, the nucleotide sequence encodinghuman TLR8 is operatively linked to a promoter and/or other regulatoryregions. In some embodiments, the promoter and/or other regulatoryregions are those of human TLR8. In some embodiments, the human TLR8 isexpressed in a similar expression pattern in the transgenic animal ashuman TLR8 is expressed in humans. In some embodiments, the levels ofexpression of human TLR8 in the transgenic animal is similar to thelevel of expression of human TLR8 in humans. In some embodiments, thetransgenic animal is predisposed to development of inflammation in oneor more organs (e.g., pancreas, kidney, liver, joints, reproductivetissue, etc.). In some embodiments, the inflammation comprises anautoimmune disease (e.g., pancreatitis, nephritis, hepatitis, rheumatoidarthritis, diabetes, diabetes-related disorder, reproductive disease,etc.). In some embodiments, the present disclosure provides a cellobtained from the transgenic animal described herein, wherein the humanTLR8 is expressed in the cell. In some embodiments, the cell is ahematopoietic cell. In some embodiments, the hematopoeitic cell is amonocyte. Provided herein are also methods of screening candidateagents, the methods comprising: administering a candidate agent to thetransgenic animal; and determining the effect of the candidate agent onthe animal (e.g., as compared to a control such as untreated animal oran animal receiving a placebo). Some methods comprise administering acandidate agent to the transgenic animal; and determining the effect ofthe candidate agent on a TLR8-mediated response of the animal (e.g., ascompared to a control such as an untreated animal or an animal receivinga placebo). Additionally, provided herein are methods of screeningcandidate agents, the method comprising: contacting the cell obtainedfrom the transgenic animal with a candidate agent; and determining theeffect of the candidate agent on the cell (e.g., as compared to acontrol such as an untreated or mock-treated cell). Some methodscomprise contacting the cell obtained from the transgenic animal with acandidate agent; and determining the effect of the candidate agent onthe cell (e.g., as compared to a control such as untreated ormock-treated cell). In some embodiments, the effect comprises inhibitionof the TLR8-mediated response (e.g., reduction of the response to lessthan 50, 60, 70, 75, 80, 85, 90 or 95% of that observed in the absenceof the candidate agent). In alternative embodiments, the effectcomprises stimulation of the TLR8-mediated response (e.g., elevation ofthe response to at least 150, 160, 170, 180, 190 or 200% of thatobserved in the absence of the candidate agent). In some, theTLR8-mediated response is evidenced by a change in TLR8-mediatedcytokine production, cell proliferation, and/or cell surface markerexpression. A TLR8-mediated response is one that is stimulable by aTLR7/8 agonist such as R848 and CL075 or a TLR8 agonist such as a TLR8ligand stabilized immunomodulatory RNA (e.g., SEQ ID NO:3).TLR8-mediated responses are assessed by measuring expression of acytokine or a cell surface marker. Suitable cytokines are selected frombut not limited to TNF-α, IFN-α, IFN-β, IFN-γ, IL-1α, IL-1β, IL-6, IL-8,IL-10, IL-12, IL-23, IP-10, MIP-1, and MCP-1. Suitable cell surfacemolecules are selected from but not limited CD40, CD80, CD86, ICAM-1,ICAM-2, ICAM-3, and CCR7. In some embodiments, the cytokine comprisesone or more of the group consisting of TNF, IL-12, IL-6, MIP-1α, IFNγ,IP-10, IL-1α, and IL-1β. In some embodiments, the candidate agent is anantibody. In some embodiments, the candidate agent is a small molecule.In some preferred embodiments, the candidate agent is a polynucleotide.In some preferred embodiments, the TLR8-mediated cytokine productioncomprises production of one or more of the group consisting of TNF,IL-12, IL-6, MIP-1α, IFNγ, IP-10, IL-1α, and IL-1β.

Moreover, the present disclosure provides methods for screening, and/oridentifying, TLR8 modulators, the method comprising: providing acandidate agent to a cell culture, wherein cells of the cell culturecomprises cells derived from a TLR8 transgenic animal which comprises anucleotide sequence encoding human TLR8; and determining the effect ofthe candidate agent on the cell culture (as compared to a control suchas untreated or mock-treated cell culture). Further, provided aremethods of screening for, and/or identifying, TLR8 modulators, themethod comprising: providing a candidate agent to a cell culture,wherein cells of the cell culture are obtained or prepared from atransgenic animal described herein which comprise a nucleotide sequenceencoding human TLR8; and determining the effect of the candidate agenton the cell culture (as compared to a control such as untreated ormock-treated cell culture). In some embodiments, the cells arehematopoietic cells. In some embodiments, the cells are monocytes. Insome embodiments, the effect is inhibition of cytokine production, cellproliferation and/or cell surface molecule expression. In someembodiments, the effect is stimulation of cytokine production, cellproliferation and/or cell surface molecule expression. Suitablecytokines are selected from but not limited to TNF-α, IFN-α, IFN-β,IFN-γ, IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-12, IL-23, IP-10, MIP-1, andMCP-1. Suitable cell surface molecules are selected from but not limitedCD40, CD80, CD86, ICAM-1, ICAM-2, ICAM-3, and CCR7. In some embodiments,the cytokine comprises one or more of the group consisting of TNF,IL-12, IL-6, MIP-1α, IFNγ, IP-10, IL-1α, and IL-1β. In some embodiments,the candidate agent is an antibody (e.g., antibody fragment). In someembodiments, the candidate agent is a small molecule. In someembodiments, the candidate agent is a polynucleotide. In someembodiments, the polynucleotide comprises a TLR8 immunoregulatorysequence (IRS). In some embodiments, the polynucleotide comprises a TLR8immunostimulatory sequence (ISS). In some embodiments, the candidateagent is an antagonist. In some embodiments, the candidate agent is anagonist.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the level of expression of human TLR8 in purified humansubsets:

-   -   plasmacytoid dendritic cells (PDC), monocytes, myeloid dendritic        cells (MDC), CD4+ T-cells, CD8+ T-cells, and neutrophils.

FIG. 2A-B shows the effect of 200, 100, and 20 μg/mL of TLR7 orTLR8-stimulating RNA polynucleotides (PN) (SEQ ID NO:2 and SEQ ID NO:3,respectively) or medium alone on hIL-6 and hTNF production (pg/mL) byhuman peripheral blood mononuclear cells (PBMC) and monocytes.

FIG. 3A-C shows the effect of 100 μg/mL of TLR7-stimulating RNA PN (SEQID NO:2), 100 μg/mL of TLR8-stimulating RNA PN (SEQ ID NO:3), or mediumalone on hIL-6, hTNF and hIFN-α production (pg/mL) by human PDC.

FIG. 4A-D shows the effect of 100 μg/mL of TLR7-stimulating RNA PN (SEQID NO:2), 100 μg/mL of TLR8-stimulating RNA PN (SEQ ID NO:3), or mediumalone on mIL-12 production (pg/mL) and mTNF production (pg/mL) by mousesplenocytes and mouse PBMC, respectively.

FIG. 5 shows the TLR8 BAC construct used to produce the TLR8 transgenicmice.

FIG. 6A-C shows the correlation between survival and the level of humanTLR8 gene expression in chimeric mice, and the elevated level ofexpression of TNF, IL-6, IL-17, IL-12p40, CCL-2 and IP10 in sera ofTLR8Tg mice. FIG. 6A shows the survival curve of human TLR8 chimericmice from clone 6 (number of mice=14), clone 23 (number of mice=8),clone 12 (number of mice=18), and clone 8 (number of mice=18). FIG. 6Bshows the TLR8 expression levels as relative CT in these mice. FIG. 6Cshows the elevated level of inflammatory cytokines in serum of TLR8Tgmice from clone 12 (n=15) as compared to control, wild-type C57BL/6 mice(n=18).

FIG. 7A-F shows upregulation of expression of inflammatory cytokinesIFN-γ, IL12-p40, TNF-α, IP-10, IL-1α, IL-1β by relative CT in thepancreas from chimeric mice from clone 6, 12 and 23 (total number ofmice=6) compared to wild-type mice C57BL/6 (total number of mice=6).FIG. 7G-7I shows the titers of anti-nuclear antibody (ANA), anti-doublestranded DNA (dsDNA) antibody and anti-ribonucleoprotein (RNP) antibodyin the serum of TLR8TgCL12 (n=27) and age-matched controls (n=37).

FIG. 8A-D shows the levels of CD80, GITRL, CD40, and OX40L as determinedby FACS analyses of myeloid dendritic cells (MDC) from chimericTLR8TGCL23 mice as compared to wild-type C57BL/6 mice.

FIG. 9 shows the percentage survival per day post-bone marrow transplantof mice receiving bone marrow from TLR8 chimeric mice from clones 6, 12and 23.

FIG. 10A-B shows example of dot blot results of FACS analyses of T-cellsfrom the spleen and blood of mice transplanted with bone marrow fromchimeric TLR8TGCL12 mice. Examples of dot blots for both CD4 T-cells(FIG. 10A) and for CD8 T-cells (FIG. 10B) are provided.

FIG. 11A-L shows cumulative data of FACS analyses of T-cells from thespleen and blood of mice transplanted with bone marrow from chimericTLR8TGCL12 mice.

FIG. 12A-E shows expression of MHC Class II, CD80, GITRL, OX40L, andPDL-1, respectively, as determined by FACS analyses of dendritic cellsof mice transplanted with human TLR8 transgenic mouse bone marrow.

FIG. 13A-C shows activation of TLR8 expressed from the human TLR8transgene in blood cells in TLR8TGCL8 mice as compared to C56BL/6wild-type mice upon activation with 200 μg/mL or 100 μg/mL ofTLR8-stimulating RNA PN (SEQ ID NO:3), or media alone as assayed bymeasuring levels of mIL-12 (pg/mL), mTNF-α (pg/mL), and mIL-6 (pg/mL),respectively.

FIG. 14A-B shows activation of TLR8 expressed from the human TLR8transgene in bone marrow (BM) cells from TLR8TGCL8 mice upon activationwith 100 μg/mL or 50 μg/mL of a TLR8-specific ligand (SEQ ID NO:3) asassayed by measuring levels of mTNF-α (pg/mL) and mIL-6 (pg/mL),respectively.

FIG. 15A-C shows that mice over expressing human TLR8 spontaneouslydevelop arthritis. Chimeric mice from clone 12 (n=6) and C57BL/6 CTLRanimals (n=6) were euthanized 90 days after birth. All the hTLR8 animalsexhibited signs of swelling in forelimbs and hindlimbs. Paws and jointswere fixed, sectioned and stained with toluene blue. FIG. 15A provides arepresentative section of a joint from WT (C57BL/6 CTLR) mice. FIG. 15Bprovides a representative section of a joint from a TLR8Tg mouse. Thehistological changes, degree of inflammation and cartilage damage wereevaluated by a pathologist in a blinded fashion and scored 1 to 5 asfollows: 1=minimal, 2=mild, 3=moderate, 4=marked, 5=severe. Two paws andtwo ankles were evaluated for each mouse, and the scores were summed toobtain the histological disease score shown in FIG. 15C.

FIG. 16A-B shows the cumulative score and the percentage incidence ofsymptoms with a clinical score of greater than or equal to four,respectively, using a collagen-induced rheumatoid arthritis (CIA) modelfor wild-type mice (C57BL/6) and TLR8 transgenic mice (TLR8TGCL8) indays after first collagen injection. FIG. 16C-E shows that hTLR8, F4/80and TNF were significantly elevated in joints of hTLR8 transgenic mice(TLR8Tg clone 8) as compared to wild-type mice (C57BL/6). FIG. 16F-Hshows hTLR8, F4/80 and TNF expression relative to the disease score orlevel of hTLR8 expression respectively. Clearly, the levels of F4/80 andTNF were directly correlated with the level of hTLR8 in the transgenicmice. Likewise, the levels of proinflammatory cytokines (IL-1beta,IL-1alpha, IL-6, IL-18, IP-10, and MMP-9) were found to be directlycorrelated with hTLR8 expression.

FIG. 17 shows a significant increase in blood glucose level (mg/dL) inmice over-expressing human TLR8 (C57BL/6-SJL WT mice transplanted withTLR8TGCL12 bone marrow cells) compared to C57BL/6-SJL WT micetransplanted with WT C57BL/6 bone marrow cells.

FIG. 18A shows that human TLR8 is expressed in monocytes, neutrophilsand dendritic cells of hTLR8Tg mice. The expression pattern of huTLR8 inleukocyte subsets from these mice is very similar to its expression inhuman leukocytes. FIG. 18B-D shows that hTLR8 is functional in hTLR8Tgmice. Briefly, PBMC from hTLR8Tg chimeras, but not from wild-type miceproduced IL-12 and TNF when stimulated with an RNA-based TLR8 agonist,ORN-8L (FIG. 18B-C). Similarly, elevated levels of IL-12 were detectedin the serum from hTLR8Tg mice, but not from wild-type mice, which wereinjected intravenously with an RNA-based TLR8 agonist (ORN-8L) or asmall molecule-based TLR8 agonist (CL075).

FIG. 19A-C shows that hTLR8Tg mice develop lymphopenia (decrease innumber of circulating lymphocytes), granulocytosis (increase in numberof granulocytes, which are primarily neutrophils) and monocytosis(increase in number of monocytes). Cell number was assessed inperipheral blood (FIG. 19A) and in spleens of hTLR8Tg chimeras orage-matched control C57BL/6 mice (FIG. 19B). There is a significantdecrease in circulating lymphocytes paralleled by an increase ingranulocytes and monocytes in the blood of the hTLR8-expressing mice ascompared to WT mice CTRL (FIG. 19C).

FIG. 20A-C shows that T lymphocytes from mice over-expressing human TLR8produce more TNF and IFN-gamma as compared to T lymphocytes from wildtype mice.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are hTLR8 transgenic animals, and cells therefrom thatexpress human TLR8. Also provided are methods of screening for and/oridentifying TLR8 modulators using the hTLR8 transgenic animals or cellsexpressing human TLR8.

Unless otherwise indicated, reference to an agent can include thecompound in any pharmaceutically acceptable form, including any isomer(e.g., diastereomer or enantiomer), salt, solvate, polymorph, and thelike. In particular, if a compound is optically active, reference to thecompound can include each of the compounds enantiomers as well asracemic mixtures of the enantiomers.

DEFINITIONS

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide”include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA),single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), modifiedpolynucleotides and polynucleosides or combinations thereof. Thepolynucleotide can be linearly, branched, or circularly configured, orthe polynucleotide can contain one or more linear, branched, and/orcircular segments. Polynucleotides are polymers of nucleosides joined,generally, through phosphodiester linkages, although alternate linkages,such as phosphorothioate esters may also be used in polynucleotides. Anucleoside consists of a purine (adenine (A) or guanine (G) orderivative thereof) or pyrimidine (thymine (T), cytosine (C) or uracil(U), or derivative thereof) base bonded to a sugar. The four nucleosideunits (or bases) in DNA are called deoxyadenosine, deoxyguanosine,thymidine, and deoxycytidine. A nucleotide is a phosphate ester of anucleoside.

The term “peptide” includes polypeptides that are of any length andcomposition to affect a biological response, e.g., antibody productionor cytokine activity whether or not the peptide is a hapten. The term“peptide” further includes modified amino acids (whether or notnaturally or non-naturally occurring), such modifications including, butnot limited to, phosphorylation, glycosylation, pegylation, lipidizationand methylation.

The terms “promoter element” or “promoter” refer to a DNA regulatoryregion capable of binding an RNA polymerase in a cell (e.g., directly orthrough other promoter-bound proteins or substances) and initiatingtranscription of a coding sequence. A promoter sequence is, in general,bounded at its 3′ terminus by the transcription initiation site andextends upstream (5′ direction) to include the minimum number of basesor elements necessary to initiate transcription at any level. Within thepromoter sequence may be found a transcription initiation site(conveniently defined, for example, by mapping with nuclease S1), aswell as protein binding domains (consensus sequences) responsible forthe binding of RNA polymerase. The promoter may be operably associatedwith other expression control sequences, including enhancer andrepressor sequences.

The term “vector” refers to a nucleic acid assembly capable oftransferring gene sequences to target cells (e.g., viral vectors,non-viral vectors, particulate carriers, and liposomes). The term“expression vector” refers to a nucleic acid assembly containing apromoter that is capable of directing the expression of a sequence orgene of interest in a cell. Vectors typically contain nucleic acidsequences encoding selectable markers for selection of cells that havebeen transfected by the vector. Generally, “vector construct,”“expression vector,” and “gene transfer vector,” refer to any nucleicacid construct capable of directing the expression of a gene of interestand which can transfer gene sequences to target cells. Thus, the termincludes cloning and expression vehicles, as well as viral vectors.

The term “wild-type” refers to a nucleic acid, protein, and/or animal(e.g., mouse) that has the characteristics of that nucleic acid,protein, and/or animal (e.g., mouse) when isolated from a naturallyoccurring source. A wild-type nucleic acid, protein, and/or animal(e.g., mouse) is that which is most frequently observed in a populationand is thus arbitrarily designed the “normal” or “wild-type” form ofthat molecule. In contrast, the term “modified” or “mutant” refers to anucleic acid, protein, and/or animal (e.g., mouse) that displaysmodifications in sequence and/or functional properties (i.e., alteredcharacteristics) when compared to the wild-type nucleic acid, protein,and/or animal (e.g., mouse).

The term “agonist” is used in the broadest sense and refers to amolecule that can elevate or stimulate an induced cellular activity

The term “antagonist” or “inhibitor” is used in the broadest sense, andrefers to a composition that can reduce or inhibit an induced cellularactivity.

The term “antibody” is used in the broadest sense and specificallycovers, for example, monoclonal antibodies (including agonist,antagonist, and neutralizing antibodies), antibody compositions withpolyepitopic specificity, polyclonal antibodies, single chainantibodies, and fragments of antibodies as long as they exhibit thedesired biological or immunological activity. The term “immunoglobulin”(Ig) is used interchangeably with antibody herein.

The term “small molecule” as used herein refers to a low molecularweight organic compound (e.g., not a polymer). In some embodiments, thesmall molecule has a molecular weight of less than 2000, 1600, 800, or400 daltons.

The term “immunostimulatory” or “stimulating an immune response” as usedherein includes stimulation of cell types that participate in immunereactions and enhancement of an immune response to a specific antigenicsubstance. An immune response that is stimulated by an immunostimulatorynucleic acid is generally a “Th1-type” immune response, as opposed to a“Th2-type” immune response. Th1-type immune responses are normallycharacterized by “delayed-type hypersensitivity” reactions to an antigenand activated macrophage function and can be detected at the biochemicallevel by increased levels of Th1-associated cytokines such as IFN-γ,IL-2, IL-12, and TNF-α. Th2-type immune responses are generallyassociated with high levels of antibody production, especially IgEantibody production and enhanced eosinophils numbers and activation, aswell as expression of Th2-associated cytokines such as IL-4, IL-5 andIL-13.

The term “immunoregulatory compound” or “IRC”, as used herein, refers toa molecule which has immunoregulatory activity and which comprises anucleic acid moiety comprising an IRS. The IRC may consist of a nucleicacid moiety that comprises more than one IRS or consists of an IRS. TheIRC may comprise a modified and/or unmodified IRS. The IRC may consistof a polynucleotide (a “polynucleotide IRC”) or it may compriseadditional moieties. Accordingly, the term IRC includes compounds whichincorporate one or more nucleic acid moieties, at least one of whichcomprises an IRS, covalently linked to a non-nucleotide spacer moiety.

The term “immunoregulatory sequence” or “IRS”, as used herein, refers toa nucleic acid sequence that inhibits and/or suppresses a measurableimmune response as measured in vitro, in vivo, and/or ex vivo. The term“immunoregulatory sequence” or “IRS”, as used herein, refers to bothnucleic acid sequences that comprise a modification (i.e., modified IRS)as well as nucleic acids which do not comprise a modification (i.e.,unmodified IRS).

The term “TLR8 modulator” is used in the broadest sense, and includesTLR8 agonists and antagonists.

The term “cells,” as used herein, is understood to refer not only to theparticular subject cell, but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not in fact be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

The term “transfection” refers to the uptake of DNA by a cell. A cellhas been “transfected” when exogenous (i.e., foreign) DNA has beenintroduced inside the cell membrane. Transfection can be eithertransient (i.e., the introduced DNA remains extrachromosomal and isdiluted out during cell division) or stable (i.e., the introduced DNAintegrates into the cell genome or is maintained as a stable episomalelement).

“Stimulation” of a response or parameter includes eliciting and/orenhancing that response or parameter when compared to otherwise sameconditions except for a condition or parameter of interest, oralternatively, as compared to another condition. For example,“stimulation” of an immune response means an increase in the response,which can arise from eliciting and/or enhancement of a response.Similarly, “stimulation” of production of a cytokine (such as IL-1α,IL-1β, IL-6, and/or TNF-α) or “stimulation” of cell type (such as CTLs)means an increase in the amount or level of cytokine or cell type.

“Suppression” or “inhibition” of a response or parameter includesdecreasing that response or parameter when compared to otherwise sameconditions except for a condition or parameter of interest, oralternatively, as compared to another condition.

“Correlate” or “correlating” as used herein refer to comparing, in anyway, the performance and/or results of a first analysis or protocol withthe performance and/or results of a second analysis or protocol. Forexample, one may use the results of a first analysis or protocol todetermine whether a second analysis or protocol should be performed.With respect to the embodiment of gene expression analysis or protocol,one may use the results of the gene expression analysis or protocol todetermine whether a specific therapeutic regimen should be performed.

The term “innate immune response” or “innate immunity” as used hereinincludes a variety of innate resistance mechanisms by which a cell orindividual recognizes and responds to the presence of a pathogen. Asused herein, an “innate immune response” includes the intracellular andintercellular events and reactions that occur when the cell recognizespathogen associated molecular patterns or signals. Cellular receptorsactive in an innate immune response include a family of Toll-likereceptors (TLRs) and microbial ligands have been identified for severalTLRs, as described herein.

The term “individual” refers to a mammal, including humans. Anindividual includes, but is not limited to, human, bovine, horse,feline, canine, rodent, or primate.

The term “animal” is used herein to include all vertebrate andinvertebrate animals, except humans. It also includes an individualanimal in all stages of development, including embryonic and fetalstages. A “transgenic animal” is an animal containing one or more cellsbearing genetic information received, directly or indirectly, bydeliberate genetic manipulation at a subcellular level, such as bymicroinjection or infection with recombinant virus. This introduced DNAmolecule may be integrated within a chromosome, or it may beextra-chromosomally replicating DNA.

The term “germ cell-line transgenic animal” refers to a transgenicanimal in which the genetic information was introduced into a germ linecell, thereby conferring the ability to transfer the information tooffspring. If such offspring in fact possess some or all of thatinformation, then they, too, are transgenic animals.

“Adjuvant” refers to a substance which, when added to an immunogenicagent such as antigen, nonspecifically enhances or potentiates an immuneresponse to the agent in the recipient host upon exposure to themixture.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration refers to treatment that is notconsecutively and/or continuously done without interruption, but ratheris cyclic in nature.

An “effective amount” of an agent disclosed herein is an amountsufficient to carry out a specifically stated purpose. An “effectiveamount” may be determined empirically and in a routine manner, inrelation to the stated purpose.

A “growth inhibitory amount” as used herein is an amount capable ofinhibiting the growth of a cell, especially tumor, e.g., cancer cell,either in vitro or in vivo. A “growth inhibitory amount” for purposes ofinhibiting neoplastic cell growth may be determined empirically and in aroutine manner.

The term “therapeutically effective amount” refers to an agent effectiveto “treat” a disease or disorder in a subject or mammal. In the case ofcancer, the therapeutically effective amount of the agent may reduce thenumber of cancer cells: reduce the tumor size; inhibit (i.e., slow tosome extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. See the definition herein of “treating”. To the extent the drugmay prevent growth and/or kill existing cancer cells, it may becytostatic and/or cytotoxic.

As used herein, “sample” refers to a composition which contains amolecule which is to be characterized and/or identified, for example,based on physical, biochemical, chemical, physiological, and/or geneticcharacteristics.

As used herein, and as well-understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, including clinicalresults. Beneficial or desired clinical results include, but are notlimited to, alleviation or amelioration of one or more symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, preventing spread of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Thus, “treating” or“treatment” does not require complete alleviation of signs or symptoms,and does not require a cure.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise.

It is understood that aspects and embodiments described herein include“consisting” and/or “consisting essentially of” aspects and embodiments.

Human TLR8 Transgenic Animals and Cells

Provided herein are TLR8 transgenic animals and transgenic cells, whichcomprise a nucleotide sequence encoding human TLR8. For example,provided herein are transgenic animals comprising a nucleotide sequenceencoding human TLR8, wherein the human TLR8 is expressed in thetransgenic animal.

In some embodiments, the TLR8 transgenic animal is a chimeric TLR8transgenic animal. In some embodiments, the TLR8 transgenic animal is aTLR8 transgenic animal with germ cells and somatic cells containing anucleotide sequence encoding human TLR8. In some embodiments, thenucleotide sequence encoding human TLR8 is stably integrated into thegenome of the TLR8 transgenic animal. In some embodiments, thenucleotide sequence encoding human TLR8 is extrachromosomal. In someembodiments, the extrachromosomal nucleotide sequence encoding humanTLR8 is provided as a minichromosome, yeast artificial chromosome, orbacterial artificial chromosome. In some embodiments, the TLR8transgenic animal comprises one or more copies of the nucleotidesequence encoding human TLR8. In some embodiments, the TLR8 transgenicanimal comprises more than about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 copies of the nucleotide sequence encoding humanTLR8. In some embodiments, the TLR8 transgenic animal comprises aboutany of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 copies ofthe nucleotide sequence encoding human TLR8. For example, providedherein are transgenic animals comprising a nucleotide sequence encodinghuman TLR8, wherein the human TLR8 is expressed in the transgenicanimal, wherein the TLR8 transgenic animal comprises about any of 1 to 2copies of the nucleotide sequence encoding human TLR8.

The terms “human Toll-like receptor 8,” “human TLR8,” “hTLR8,” and“CD288” as used herein refer to a protein and functional variantsthereof that bind to viral and synthetic single-stranded RNAs (e.g.,ssRNA derived from viruses, synthetic guanosine-rich ssRNA, anduridine-rich ssRNA), as well as small molecules resembling nucleicacids. The amino acid sequence of a common isoform of hTLR8 is set forthas SEQ ID NO:4. Accordingly, hTLR8 comprises the amino acid sequence ofSEQ ID NO:4 or variants having at least 90, 91, 92, 93, 94, 95, 96, 97,98 or 99% identity to SEQ ID NO:4. In particularly preferredembodiments, the “RQSYA” motif (SEQ ID NO:5) in the ectodomain of hTLR8is retained.

Human TLR8 Sequence of GENBANK Accession No. NP_619542.1 (SEQ ID NO: 4):   1 menmflqssm ltcifllisg scelcaeenf srsypcdekk qndsviaecs nrrlqevpqt  61 vgkyvteldl sdnfithitn esfqglqnlt kinlnhnpnv qhqngnpgiq snglnitdga 121 flnlknlrel llednqlpqi psglpeslte lsliqnniyn itkegisrli nlknlylawn 181 cyfnkvcekt niedgvfetl tnlellslsf nslshvppkl psslrklfls ntqikyisee 241 dfkglinitl ldlsgncprc fnapfpcvpc dggasinidr fafqnitqlr ylnlsstslr 301 kinaawfknm phlkvldlef nylvgeiasg afltmlprle ildlsfnyik gsypqhinis 361 rnfskllslr alhlrgyvfq elreddfqpl mqlpnlstin lginfikqid fklfqnfsnl 421 eiiylsenri splvkdtrqs yansssfqrh irkrrstdfe fdphsnfyhf trplikpqca 481 aygkaldlsl nsiffigpnq fenlpdiacl nlsansnaqv lsgtefsaip hvkyldltnn 541 rldfdnasal telsdlevld lsynshyfri agvthhlefi qnftnlkvin lshnniytlt 601 dkynlesksl velvfsgnrl dilwndddnr yisifkglkn ltrldlslnr lkhipneafl 661 nlpasltelh indnmlkffn wtllqqfprl elldlrgnkl lfltdslsdf tsslrtllls 721 hnrishlpsg flsevsslkh ldlssnllkt inksaletkt ttklsmlelh gnpfectcdi 781 gdfrrwmdeh lnvkiprlvd vicaspgdqr gksivslelt tcvsdvtavi lffftffitt 841 mvmlaalahh lfywdvwfiy nvclakvkgy rslstsqtfy dayisydtkd asvtdwvine 901 lryhleesrd knvllcleer dwdpglaiid nlmqsingsk ktvfvltkky akswnfktaf 961 ylalqrlmde nmdviifill epvlqhsgyl rlrgrickss ilqwpdnpka eglfwqtlrn1021 vvltendsry nnmyvdsikq y.

Thus the variants comprise homologous TLR8 amino acid sequences havingone or more deletions, additions or substitutions as long as therequisite level of sequence identity across the full length of TLR8 isachieved. Sequence identity may be determined using known programs suchas BLAST, ALIGN, and CLUSTAL using standard parameters. (See, e.g.,Altschul et al. [1990] J. Mol. Biol. 215:403-410; Henikoff et al. [1989]Proc. Natl. Acad. Sci. USA 89:10915; Karin et al. [1993] Proc. Natl.Acad. Sci USA 90:5873; and Higgins et al. [1988] Gene 73:237-244).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. Databases may also besearched using FASTA (Pearson et al. [1988] Proc. Natl. Acad. Sci. USA85:2444-2448).

In some embodiments, the TLR8 transgenic animal expresses differentiallevels of human TLR8. In some embodiments, the TLR8 transgenic animalexpresses high levels of human TLR8. In some embodiments, the highlevels of human TLR8 expression is the result of multiple copy number,the site of integration of the nucleotide sequence encoding human TLR8,and/or the promoter and/or regulatory region operably linked to thenucleotide sequence encoding human TLR8. In some embodiments, theexpression level is between a relative CT value of 10 and 10,000, forexample about any of between 100-500, 200-400, or 1500-1700. In someembodiments, the expression level is at least about a relative CT valueof any one of 10, 100, 200, 300, 400, 500, 1000, 1500, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, or 10,000. Relative CT values can beevaluated, for example, by obtaining threshold cycle (CT) values foreach gene of interest and normalizing to a housekeeping gene using theformula: relativeCT=1.8^((Avg CT Housekeeping Gene-CT Gene of Interest))*100,000. In someembodiments, the gene expression is evaluated by the average relativeCT. In some embodiments, the Avg CT housekeeping gene is the mean CT oftriplicate housekeeping gene runs and/or Avg CT Gene of Interest is themean CT of duplicate runs of the gene of interest. In some embodiments,the house keeping gene is ubiquitin, myosin, hsp90, and/or actin.

In some embodiments, the expression level is about the same or greaterthan the expression level of human TLR8 in TLR8TGCL12. In someembodiments, the expression level is about the same or greater than theexpression level of human TLR8 in TLR8TGCL6. In some embodiments, theexpression level is about the same or greater than the expression levelof human TLR8 in TLR8TGCL23. In some embodiments, the expression levelis about the same or greater than the expression of human TLR8 inTLR8TGCL8. In some embodiments, the human TLR8 is expressed in a similarexpression pattern in the transgenic animal as human TLR8 is expressedin humans. In some embodiments, the levels of expression of human TLR8in the transgenic animal is similar to the level of expression of humanTLR8 in humans. In some embodiments, the levels of expression of humanTLR8 in the transgenic animal is about any of 1, 2, 3, 4, 5-fold thelevel of expression of human TLR8 in humans. In some embodiments, thelevels of expression of human TLR8 in the transgenic animal is similarto the level of expression of mouse TLR8 in mice.

In some embodiments, the TLR8 transgenic animal has reduced survival(e.g., life span), for example, compared to a wild-type animal. Forexample, in some embodiments, the transgenic animal survives less thanabout any of 150, 140, 130, 120, 110, 100, 90, 80, 70, 60 or 50 days.For example, provided herein are transgenic animals comprising anucleotide sequence encoding human TLR8, wherein the human TLR8 isexpressed in the transgenic animal, wherein the TLR8 transgenic animalsurvives less than about 150 days. In some embodiments, the TLR8transgenic animal has survival (e.g., life span) similar orsubstantially the same as compared to a wild-type animal.

The human TLR8 gene may be of natural or artificial origin. It may begenomic DNA (gDNA), complementary DNA (cDNA), hybrid sequences orsynthetic or semi-synthetic sequences. To be expressed, the human TLR8gene should be operably linked to one or more regulatory regions.Selection of the appropriate regulatory region or regions is a routinematter, within the level of ordinary skill in the art. Regulatoryregions may be used to increase, decrease, or regulate the expression ofa gene or to designate the expression of a gene to certain tissues or tocertain stages of development. In some embodiments, the regulatoryregion increases expression of the gene or increases expression of thegene in neutrophils. The regulatory regions may comprise a promoterregion for functional transcription, as well as a region situated 3′ ofthe gene of interest, and which specifies a signal for termination oftranscription and a polyadenylation site. All these elements constitutean expression cassette.

Promoters that may be used include both constitutive promoters andregulated (inducible) promoters. The promoter may be naturallyresponsible for the expression of the nucleic acid. It may also be froma heterologous source. In particular, it may be promoter sequences ofeukaryotic or viral genes. For example, it may be promoter sequencesderived from the genome of the cell which it is desired to infect.Likewise, it may be promoter sequences derived from the genome of avirus, including the adenovirus used. The promoters of the ElA, MLP,HCMV and RSV genes and the like may be used. In addition, the promotermay be modified by addition of activating or regulatory sequences, orsequences allowing a tissue-specific or predominant expression. Thepromoter need not be a naturally occurring promoter. The promoter may bean inducible promoter. In some embodiments, the human TLR8 gene isoperably linked to the human TLR8 promoter. In some embodiments, thehuman TLR8 gene is operably linked to the human TLR8 promoter and humanTLR8 regulatory region.

Tetracycline-inducible systems can be used, as described inHickman-Davis et al. (Pediatric Respiratory Reviews 2006 7: 49). Twoindependent transgenic mouse lines are generated: (1) transactivatormice, in which a tetracycline-controlled transactivator is expressedunder the control of a tissue-specific promoter and (2) responder mice,in which expression of the DNA of interest is under the control of atetracycline-dependent promoter (a minimal RNA polymerase II promoterfused to tet operator sequences). The breeding of these two strains ofmice generates a double-transgenic mouse that responds to tetracyclineor its derivatives (doxycycline) to control expression of the transgene.There are two possible mirror image tetracycline-inducible systems. Inthe first system, the absence of doxycycline allows for transcription ofthe transgene and addition of tetracycline or its derivatives causestranscriptional downregulation (tet-OFF). In the second systemtranscription of the transgene occurs in the presence of doxycycline andtherefore removal of the activator results in transcriptionaldownregulation (tet-ON).

Additional useful promoters are the ubiquitous promoters HPRT, vimentin,actin, and tubulin; the intermediate filament promoters desmin,neurofilaments, keratin, and GFAP; the therapeutic gene promoters MDR,CFTR, and factor VIII; promoters which are preferentially activated individing cells; cytomegalovirus immediate-early; retroviral LTR,metallothionein; SV-40; Ela and MLP promoters. Tetracycline-regulatedtranscriptional modulators and CMV promoters are described in WO96/01313, U.S. Pat. Nos. 5,168,062 and 5,385,839, the contents of whichare incorporated herein by reference.

In some embodiments, a cre/loxP recombinase system is utilized forgeneration of the transgenic animals. For example, the Cre/loxPrecombinase systems described in Hickman-Davis et al. (PediatricRespiratory Reviews 2006 7: 49) can be used. For this system, thegeneration of two independent mouse lines requires: (1) mice thatcontain the target gene or gene segment flanked by two 34 bp, asymmetricnucleotide sequences (loxP) sites in the same orientation (′floxed′sequence) and (2) mice that contain a fusion transgene expressing theCre recombinase of the P1 bacteriophage. The Cre recombinase promotesrecombination by recognition of the loxP sites, and when these two mousestrains are crossed, the floxed gene is deleted and a null mutation iscreated. Cre/loxP recombinase system is also useful in the targetedmutagenesis of embryonic stem cells in vitro to create ‘clean’ mutationsthat lack a selection cassette that might interfere with generegulation, in which pluripotent stem cells containing the gene ofinterest and only one loxP site with foreign sequence are generated foruse in the creation of a transgenic mouse. Several methods have beendemonstrated for controlling Cre expression including the creation offusion proteins containing Cre and having specific ligand-bindingdomains (i.e., Cre is expressed only in the presence of a specificligand), as well as a tetracycline-inducible Cre system.

In some embodiments, the transgenic animal is a mouse or a rat. In someembodiments, the transgenic animal is a mouse. Mouse strains useful forgenerating transgenic mice include, but are not limited to CD-1® Nudemice, CD-1 mice, NU/NU mice, BALB/C Nude mice, BALB/C mice, NIH-IIImice, SCID™ mice, outbred SCID™ mice, SCID Beige mice, C3H mice, C57BL/6mice, DBA/2 mice, FVB mice, CB17 mice, 129 mice, SJL mice, B6C3F1 mice,BDF1 mice, CDF1 mice, CB6F1 mice, CF-1 mice, Swiss Webster mice, SKH1mice, PGP mice, and B6SJL mice.

The transgenic animals are produced by introducing one or more trangenesinto the germline of the transgenic animal. The methods enabling theintroduction of DNA into cells are generally available and well-known inthe art and different methods of introducing transgenes could be used.See, for example, Hogan et al. Manipulating the Mouse Embryo: ALaboratory Manual Cold Spring Harbor Laboratory, 2^(nd) edition, ColdSpring Harbor Laboratory (1994) and U.S. Pat. Nos. 5,602,299; 5,175,384;6,066,778 and 6,037,521, which are incorporated herein in theirentirety. Technology used in developing transgenic animals includepronuclear microinjection (Gordon, J. W., PNAS 77, 7380-7384 (1980) andU.S. Pat. No. 4,873,191), homologous recombination (targetedtransgenesis by transferring embryonic stem cells into blastocysts;Thompson et al., Cell 56:313-321 (1989)), RNA interference (RNAi) forsilencing of specific gene function; retrovirus gene transfer into germlines (Van der Putten et al., Proc. Nat. Acad. Sci. 82:6148-6152(1985)); electroporation of embryos (Lo, Mol. Cell. Biol. 3:1803-1814(1983)); and sperm-mediated gene transfer (Lavitrano et al., Cell57:717-723 (1989)).

Generally, the zygote is the best target for microinjection. In mice,for example, the male pronucleus reaches the size of approximately 20 μmin diameter, which allows reproducible injection of 1-2 pL of DNAsolution. The use of zygotes as a target for gene transfer has a majoradvantage. In most cases, the injected DNA will be incorporated into thehost gene before the first cleavage. Consequently, nearly all cells ofthe transgenic non-human animal will carry the incorporated transgene.Generally, this will also result in the efficient transmission of thetransgene to offspring of the founder since 50% of the germ cells willharbor the transgene. Microinjection of zygotes is one method forincorporating transgenes in practicing the invention. The pronuclearmicroinjection method of producing a transgenic animal results in theintroduction of linear DNA sequences into the chromosomes of thefertilized eggs. Bacterial Artificial Chromosome (BAC) containing thegene of interest or an alternative plasmid construct containing the geneof interest is injected into pronuclei (i.e., fertilized eggs at apronuclear state). The manipulated pronuclei are subsequently injectedinto the uterus of a pseudopregnant female. Mice generated can have oneor multiple copies of the transgene, which can be assayed by southernblot technology.

The transgenic animals can also be generated by introduction of thetargeting vectors into embryonal stem (ES) cells. ES cells are obtainedby culturing pre-implantation embryos in vitro under appropriateconditions (Evans et al., Nature 292:154-156 (1981); Bradley et al.,Nature 309:255-258 (1984); Gossler et al., PNAS 83:9065-9069 (1986); andRobertson et al., Nature 322:445-448 (1986)). Transgenes can beefficiently introduced into the ES cells by DNA transfection using avariety of methods known to the art including electroporation, calciumphosphate co-precipitation, protoplast or spheroplast fusion,lipofection and DEAE-dextran-mediated transfection. Transgenes can alsobe introduced into ES cells by retrovirus-mediated transduction or bymicro-injection. Such transfected ES cells can thereafter colonize anembryo following their introduction into the blastocoel of ablastocyst-stage embryo and contribute to the germ line of the resultingchimeric animal (reviewed in Jaenisch, Science 240:1468-1474 (1988)).Prior to the introduction of transfected ES cells into the blastocoel,the transfected ES cells can be subjected to various selection protocolsto enrich for ES cells that have integrated the transgene if thetransgene provides a means for such selection. Alternatively, PCR can beused to screen for ES cells that have integrated the transgene. Thistechnique obviates the need for growth of the transfected ES cells underappropriate selective conditions prior to transfer into the blastocoel.

Retroviral infection can also be used to introduce a transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, blastomeres may betargets for retroviral infection. Efficient infection of the blastomeresis obtained by enzymatic treatment to remove the zona pellucida. Theviral vector system used to introduce the transgene is typically areplication-defective retrovirus carrying the transgene. Transfection iseasily and efficiently obtained by culturing the blastomeres on amonolayer of virus-producing cells. Alternatively, infection can beperformed at a later stage. Virus or virus-producing cells can beinjected into the blastocoel. Most of the founder animals will be mosaicfor the transgene since incorporation occurs only in a subset of thecells which formed the transgenic non-human animal. Furthermore, thefounder animal may contain retroviral insertions of the transgene at avariety of positions in the genome; these generally segregate in theoffspring. In addition, it is also possible to introduce transgenes intothe germ line, albeit with low efficiency, by intrauterine retroviralinfection of the midgestation embryo.

Viral vectors may be used to produce a transgenic animal. Preferably,the viral vectors are replication defective, that is, they are unable toreplicate autonomously in the target cell. In general, the genome of thereplication defective viral vectors which are used lack at least oneregion which is necessary for the replication of the virus in theinfected cell. These regions can either be eliminated (in whole or inpart), be rendered non-functional by any technique known to a personskilled in the art. These techniques include the total removal,substitution (by other sequences, in particular by the inserted nucleicacid), partial deletion or addition of one or more bases to an essential(for replication) region. Such techniques may be performed in vitro (onthe isolated DNA) or in situ, using the techniques of geneticmanipulation or by treatment with mutagenic agents. Preferably, thereplication defective virus retains the sequences of its genome whichare necessary for encapsidating the viral particles.

The retroviruses are integrating viruses which infect dividing cells.The retrovirus genome includes two LTRs, an encapsidation sequence andthree coding regions (gag, pol and env). The construction of recombinantretroviral vectors has been described: see, in particular, EP 453242,EP178220, Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick,BioTechnology 3 (1985) 689, etc. In recombinant retroviral vectors, thegag, pol and env genes are generally deleted, in whole or in part, andreplaced with a heterologous nucleic acid sequence of interest. Thesevectors can be constructed from different types of retrovirus, such as,HIV, MoMuLV (“murine Moloney leukaemia virus”), MSV (“murine Moloneysarcoma virus”), HaSV (“Harvey sarcoma virus”); SNV (“spleen necrosisvirus”); RSV (“Rous sarcoma virus”) and Friend virus. Defectiveretroviral vectors are disclosed in WO95/02697.

In general, in order to construct recombinant retroviruses containing anucleic acid sequence, a plasmid is constructed which contains the LTRs,the encapsidation sequence and the coding sequence. This construct isused to transfect a packaging cell line, which cell line is able tosupply in trans the retroviral functions which are deficient in theplasmid. In general, the packaging cell lines are thus able to expressthe gag, pol and env genes. Such packaging cell lines have beendescribed in the prior art, in particular the cell line 17 (U.S. Pat.No. 4,861,719); the PsiCRIP cell line (WO90/02806) and the GP+envAm-12cell line (WO89/07150). In addition, the recombinant retroviral vectorscan contain modifications within the LTRs for suppressingtranscriptional activity as well as extensive encapsidation sequenceswhich may include a part of the gag gene. Recombinant retroviral vectorsare purified by standard techniques known to those having ordinary skillin the art. Additional means of using retroviruses or retroviral vectorsto create transgenic animals known to the art involve themicro-injection of retroviral particles or mitomycin C-treated cellsproducing retrovirus into the perivitelline space of fertilized eggs orearly embryos (WO 90/08832 (1990); Haskell and Bowen, Mol. Reprod. Dev.40:386 (1995)).

Once the founder animals are produced, they can be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic mice to produce mice homozygous fora given integration site in order to both augment expression andeliminate the need for screening of animals by DNA analysis; crossing ofseparate homozygous lines to produce compound heterozygous or homozygouslines; breeding animals to different inbred genetic backgrounds so as toexamine effects of modifying alleles on expression of the transgene andthe physiological effects of expression.

Methods of Screening for and/or Identifying TLR8 Modulators

Provided herein is a method of screening for and/or identifying TLR8modulators using the transgenic animals described herein or cellstherefrom. In some embodiments, the cells are primary cells, for examplein cell culture, obtained or prepared from the transgenic animalsdescribed herein, which comprise a nucleotide sequence encoding humanTLR8. In some embodiments, the cells are of a stable cell line derivedfrom primary cells of the transgenic animals. In some embodiments, thecells are an early passage of the primary cells. Provided herein arealso methods of screening for, and/or identifying, TLR8 modulators, themethods comprising: administering a candidate agent to the transgenicanimal or providing a candidate agent to cell(s) and/or cell cultureobtained and/or derived from the transgenic animal described herein; anddetermining the effect of the candidate agent on the transgenic animalor cell culture (e.g., as compared to an untreated or mock treatedcontrol). Further, provided are methods of screening for, oridentifying, TLR8 modulators, the method comprising: providing acandidate agent to a cell culture, wherein cells of the cell culture areobtained and/or derived from the transgenic animals described hereinwhich comprise a nucleotide sequence encoding human TLR8; anddetermining the effect of the candidate agent on the cell culture (e.g.,as compared to an untreated or mock treated control). In someembodiments, the cells are from a transgenic animal. In someembodiments, the cells are from a transgenic mouse. In some embodiments,the nucleotide sequence encoding human TLR8 results in inflammation orautoimmune diseases in one or more organs as compared with a controlnon-transgenic animal and/or elevated levels of cytokine production ascompared with a control non-transgenic animal. In some embodiments theeffect is a TLR8-mediated response, evidenced by a change inTLR8-mediated cytokine production, cell proliferation, and/or cellsurface marker expression. A TLR8-mediated response is one that isstimulable by a TLR7/8 agonist such as R848 and CL075 or a TLR8 agonistsuch as a TLR8 ligand stabilized immunomodulatory RNA (e.g., SEQ IDNO:3). TLR8-mediated responses are assessed by measuring expression of acytokine or a cell surface marker. Suitable cytokines are selected frombut not limited to TNF-α, IFN-α, IFN-β, IFN-γ, IL-1α, IL-1β, IL-6, IL-8,IL-10, IL-12, IL-23, IP-10, MIP-1, and MCP-1. Suitable cell surfacemolecules are selected from but not limited CD40, CD80, CD86, ICAM-1,ICAM-2, ICAM-3, and CCR7. In some embodiments, the cytokine comprisesone or more of the group consisting of TNF, IL-12, IL-6, MIP-1α, IFNγ,IP-10, IL-1α, and IL-1β.

In some embodiments, the candidate agent is selected and/or identifiedif the candidate agent modulates a TLR8-mediated immune response. Insome embodiments, the immune response is evidenced by level or extent ofTLR8-mediated inflammation. In some embodiments, the immune response isevidenced by level or extent of TLR8-mediated cytokine production,proliferation, marker gene production, and/or cell surface markers. Insome embodiments, the modulation of TLR8-mediated immune response isinhibition of a TLR8-mediated immune response as compared to a control.In some embodiments, the level or extent of TLR8-mediated inflammationin the transgenic animal is reduced upon administration or providing thecandidate agent compared to the control. In some embodiments, levels orextent of TLR8-mediated cytokine production, proliferation, marker geneproduction, and/or cell surface markers in the transgenic animal and/orcell(s) and/or cell culture obtained and/or derived from the transgenicanimal described herein is reduced upon administration or providing thecandidate agent compared to the control. In some embodiments, themodulation of a TLR8-mediated immune response is stimulation of aTLR8-mediated immune response as compared to a control. In someembodiments, the level or extent of TLR8-mediated cytokine production,proliferation, marker gene production, and/or cell surface markers inthe transgenic animal and/or cell(s) and/or cell culture obtained and/orderived from the transgenic animal described herein is increased uponadministration or providing the candidate agent compared to the control.

The control may be cells and/or animals, which do not express humanTLR8. In some embodiments, the control may be untreated or mock-treatedtransgenic cells and/or animals, that express human TLR8. In someembodiments, the effect of the TLR8 modulator on modulating immuneresponses is compared to its effect in a non-transgenic animal and/orcell(s) and/or a cell culture obtained and/or derived from anon-transgenic animal. In some embodiments, the effect of the TLR8modulator on modulating immune responses is compared to the effect ofthe TLR8 modulator in an animal and/or cell(s) and/or a cell cultureobtained and/or derived from the transgenic animal described herein thatdo not express human TLR8 or express a different level of human TLR8. Insome embodiments, the effect of the TLR8 modulator on modulating immuneresponses is compared to the effect of a known TLR8 modulator in a TLR8transgenic animal and/or cell(s) and/or a cell culture obtained and/orderived from the transgenic animal described herein. In someembodiments, the effect of the TLR8 modulator on modulating immuneresponses is compared to the effect of an agent which does not affecthuman TLR8 in a TLR8 transgenic animal and/or cell(s) and/or a cellculture obtained and/or derived from the transgenic animal describedherein.

In some embodiments, the TLR8 transgenic animal is a chimeric TLR8transgenic animal. In some embodiments, the TLR8 transgenic animal is aTLR8 transgenic animal with germ cells and somatic cells containing anucleotide sequence encoding human TLR8. In some embodiments, thenucleotide sequence encoding human TLR8 is stably integrated into thegenome of the TLR8 transgenic animal. In some embodiments, thenucleotide sequence encoding human TLR8 is extrachromosomal. In someembodiments, the extrachromosomal nucleotide sequence encoding humanTLR8 is provided as a minichromosome, yeast artificial chromosome, orbacterial artificial chromosome.

For example, provided herein are methods of screening for and/oridentifying TLR8 modulators, the method comprising administering acandidate agent to a transgenic animal having a genome comprising astably integrated transgene encoding human TLR8; wherein the transgeneresults in inflammation in one or more organs (e.g., pancreas, kidney,liver, joints, reproductive tissue, etc.) or an autoimmune disease(e.g., pancreatitis, nephritis, hepatitis, rheumatoid arthritis,diabetes, diabetes-related disorder, reproductive disease, etc.) ascompared with a control non-transgenic animal (or elevated levels ofcytokine production as compared with a control non-transgenic animal);and determining the effect of the candidate agent on the inflammation orthe autoimmune disease of the transgenic animal. Also, for example,provided herein are methods of screening for and/or identifying TLR8modulators, the method comprising administering a candidate agent to atransgenic animal having an extrachromosomal nucleotide sequencecomprising a transgene encoding human TLR8; wherein the transgeneresults in inflammation in one or more organs (e.g., pancreas, kidney,liver, joints, reproductive tissue, etc.) or an autoimmune disease(e.g., pancreatitis, nephritis, hepatitis, rheumatoid arthritis,diabetes, diabetes-related disorder, reproductive disease, etc.) ascompared with a control non-transgenic animal (or elevated levels ofcytokine production as compared with a control non-transgenic animal);and determining the effect of the candidate agent on the inflammation orthe autoimmune disease of the transgenic animal.

Further, for example, provided herein are methods of screening forand/or identifying TLR8 modulators, the methods comprising incubating atransgenic animal cell culture with a candidate agent, the transgenicanimal cell culture being derived from a parent transgenic animal, cellsof the culture comprising a stably integrated transgene encoding humanTLR8; and determining the effect of the candidate agent on the cellculture.

Provided herein are also methods of screening for and/or identifyingTLR8 modulators, the methods comprising: incubating a transgenic animalcell culture with a candidate agent, the transgenic animal cell culturebeing derived from a parent transgenic animal, cells of the culturecomprising an extrachromosomal nucleotide sequence comprising atransgene encoding human TLR8; and determining the effect of thecandidate agent on the cell culture.

Further provided herein are methods of screening for and/or identifyingTLR8 modulators, the methods comprising incubating a transgenic animalcell culture with a candidate agent, the transgenic animal cell culturebeing derived from a parent transgenic animal, cells of the culturecomprising a stably integrated transgene encoding human TLR8; whereinthe transgene results in inflammation in one or more organs (e.g.,pancreas, kidney, liver, joints, reproductive tissue, etc.) or anautoimmune disease (e.g., pancreatitis, nephritis, hepatitis, rheumatoidarthritis, diabetes, diabetes-related disorder, reproductive disease,etc.) as compared with a control non-transgenic animal (or elevatedlevels of cytokine production as compared with a control non-transgenicanimal cell culture); and determining the effect of the candidate agenton the cell culture.

Provided herein is a method of screening for and/or identifying TLR8modulator, the method comprising incubating a transgenic animal cellculture with a candidate agent, the transgenic animal cell culture beingderived from a parent transgenic animal, cells of the culture comprisingan extrachromosomal nucleotide sequence comprising a transgene encodinghuman TLR8; wherein the transgene results in inflammation in one or moreorgans (e.g., pancreas, kidney, liver, joints, reproductive tissue,etc.) or an autoimmune disease (e.g., pancreatitis, nephritis,hepatitis, rheumatoid arthritis, diabetes, diabetes-related disorder,reproductive disease, etc.) in one or more organs (e.g., pancreas,kidney, liver, joints, reproductive tissue, etc.) as compared with acontrol non-transgenic animal (or elevated levels of cytokine productionas compared with a control non-transgenic animal cell culture); anddetermining the effect of the candidate agent on the cell culture.

In some embodiments, the TLR8 modulator is a TLR8 agonist or antagonist.In some embodiments, the TLR8 modulator is an anti-TLR8 antibody. Insome embodiments, the TLR8 modulator is a TLR8 agonist or antagonistantibody. In some embodiments, the TLR8 modulator comprises apolynucleotide. In some embodiments, the polynucleotide comprises a TLR8immunoregulatory sequence (IRS). In some embodiments, the polynucleotidecomprising a TLR8 IRS further is not capable of modulating TLR7 and/orTLR9 and/or does not comprise (i.e., lacks) a TLR7 and/or TLR9 IRSsequence. In some embodiments, the polynucleotide comprising a TLR8 IRSfurther is capable of modulating TLR7 and/or TLR9 and/or comprises aTLR7 and/or TLR9 IRS sequence. In some embodiments, the TLR8 modulatordoes not comprise (i.e., lacks) an immunostimulatory sequence. Forexample, in some embodiments, the TLR8 modulator does not comprise(i.e., lacks) a CG sequence or TCG sequence wherein the C isunmethylated. In some embodiments, the TLR8 modulator is not (i.e.,excludes) an antisense oligonucleotide and/or does not operate by a RNAipathway. In some embodiments, the TLR8 modulator is not (i.e., excludes)a microRNA and/or siRNA. In some embodiments, the TLR8 modulator is anantisense oligonucleotide. In some embodiments, the TLR8 modulator is asmall molecule.

In some embodiments, the effect of the TLR8 modulator on modulatingimmune responses is determined by measuring TLR8-mediated cytokineproduction, proliferation, marker gene production, and/or cell surfacemarkers. In some embodiments, the effect on modulating immune responsesis inhibition of TLR8. Inhibition of a TLR response, e.g., a TLR8response, includes, but is not limited to, inhibition at the receptorsite, e.g., by preventing or blocking effective ligand-receptor binding,and inhibition of the downstream signal pathway, e.g., after effectiveligand-receptor binding.

The concentration of the candidate agent being assayed by the abovemethods may range, for example, from about 0.001 μM to about 100 μM,although in some embodiments the assay may be performed with a testcompound present in concentrations outside this range. In someembodiments, the concentration of the candidate agent is 0.001 μM, 0.01μM, 0.1 μM or 1.0 μM or greater. In some embodiments, the concentrationof the candiate agent is 100 μM, 10 μM, 1.0 μM or 0.1 μM or less. Thecell culture may be incubated with the test compound, for example, fromabout 10 minutes to about 24 hours, although in some cases theincubation period may be outside this range. The density of cellsincubated with the compound to be tested may be, for example, from about1×10⁴ to about 1×10⁷ cells/ml, although in some embodiments the assaymay be performed using a cell culture having a cell density outside thisrange.

In some embodiments, cytokine levels are determined using a commerciallyavailable ELISA assay. In other embodiments, cytokine levels aredetermined using such techniques as, for example, antibody detection andquantitation (e.g., flow cytometry, western blotting,immunohisto/cytochemistry, proteome array assays), and bioassays (e.g.,L929 cytotoxicity assay where the amount of cell death is directlyproportional to the amount of TNF-α in the sample). See, e.g., CurrentProtocols in Immunology, John Wiley and Sons, Inc. (2001).

Many different cytokines and/or markers can be assayed in the methodsdescribed above. Suitable measurable cytokines include, but are notlimited to one or more of TNF-α, IFN-α, IFN-β, IFN-γ, IL-1α, IL-1β,IL-6, IL-8, IL-10, IL-12, IL-23, IP-10, MIP-1, and MCP-1. In somepreferred embodiments, the cytokine is selected from the groupconsisting of TNFα, IL-12, IL-6, IFNγ, IP-10, IL-1α, and IL-1β. Suitablemeasurable cell surface markers include co-stimulatory markers (e.g.,CD40, CD80, CD86), intercellular adhesion molecules (e.g., ICAM-1,ICAM-2, or ICAM-3), and maturation markers such as, for example, CCR7.

Also provided by the present disclosure are kits comprising thetransgenic mice or cells derived therefrom, and instructions for use ofthe mice to screen for and/or to identify a TLR8 modulators. In someembodiments, the kits further comprise a TLR8 agonist. In someembodiments, the kits further comprise a TLR8 antagonist.

Candidate Agents

Suitable candidate agents to screen for TLR8 modulatory activityinclude, but are not limited to, polynucleotides (e.g., singe or doublestranded nucleic acids), polypeptides (e.g., antibodies or antibodyfragments), antisense oligonucleotides, and small molecules (e.g.,organic compounds having a molecular weight of less than 2000, 1600,800, or 400 daltons).

In some embodiments, the TLR8 modulatory activity is TLR8 agonism (e.g.,stimulation). In other embodiments, the TLR8 modulatory activity is TLR8antagonism (e.g., inhibition). Suitable agonist molecules includepolynucleotides comprising a TLR8 immunostimulatory sequence (ISS),agonist antibodies or antibody fragments, fragments or amino acidsequence variants of native polypeptides, peptides, and small molecules.Suitable antagonist molecules include polynucleotides comprising a TLR8immunoregulatory sequence (IRS), antagonist antibodies or antibodyfragments, fragments or amino acid sequence variants of nativepolypeptides, peptides, antisense oligonucleotides, and small molecules.

Antibodies

In some embodiments, the TLR8 modulator is an anti-TLR8 antibody. Insome embodiments, the TLR8 modulator is an antibody, which can reduceand/or inhibit a TLR8-induced cellular activity disclosed herein. Insome embodiments, the TLR8 modulator comprises an antibody, which cancause and/or enhance a TLR is an agonist antibody. In some embodiments,the anti-TLR8 antibody is an antagonist antibody.

In some embodiments, the TLR8 antibody directly binds to TLR8.Alternatively, an antibody may combine with TLR8 indirectly by, forexample, (a) forming a complex with another molecule that directly bindsto TLR8, or (b) otherwise causing the modification of another compoundso that the other compound directly binds to TLR8. In some embodiments,the anti-TLR8 antibody specifically binds TLR8, for example,specifically binds TLR8, but not TLR7 and/or TLR9. In some embodiments,the binding of the receptor is measured in a cell-free assay.

An antibody “which binds” TLR8 is one that binds TLR8 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent, e.g., in targeting a cell expressing TLR8, and doesnot significantly cross-react with other proteins. In such embodiments,the extent of binding of the antibody to a “non-target” protein will beless than about 10% of the binding of the antibody to its particularTLR8 protein as determined by fluorescence activated cell sorting (FACS)analysis or radioimmunoprecipitation (RIA). An antibody that“specifically binds to” or is “specific for” TLR8 polypeptide or anepitope on TLR8 is one that binds to that TLR8 polypeptide or epitope onTLR8 without substantially binding to any other polypeptide orpolypeptide epitope.

In some embodiments the anti-TLR8 antibody is a monoclonal antibody, apolyclonal antibody, a chimeric antibody, a humanized antibody, a humanantibody, an intact antibody, a single chain antibody, or an antibodyfragment. In some embodiments, the antibody fragment is Fab, Fab′,F(ab′)2, Fv fragments; diabodies; or linear antibody. In someembodiments, the anti-TLR8 antibody or fragment thereof is isolated(e.g., identified and separated and/or recovered from a component of itsnatural environment).

The anti-TLR8 antibody may be from any of the five classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chainsdesignated α, δ, ε, γ, and μ, respectively. The γ and α classes arefurther divided into subclasses on the basis of relatively minordifferences in CH sequence and function, e.g., humans express thefollowing subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

In some embodiments, the anti-TLR8 antibody suppresses and/or reducesTLR8-mediated cytokine production. In some embodiments, the anti-TLR8antibody suppresses and/or reduces extent and/or levels of TLR8-mediatedcytokine production as compared to, for example, extent and/or levels ofcytokine produced during untreated conditions (e.g., media alone orbuffer alone). In some embodiments, the anti-TLR8 antibody causes and/orenhances TLR8-mediated cytokine production. In some embodiments, theanti-TLR8 antibody causes and/or enhances extent and/or levels ofTLR8-mediated cytokine production as compared to, for example, extentand/or levels of cytokine produced during untreated conditions (e.g.,media alone or buffer alone).

Small Molecules

In some embodiments, the TLR8 modulator is a TLR8 modulating chemicalcompound (e.g., small molecule). In some embodiments, the TLR8 modulatoris a TLR8 modulating chemical compound (e.g., small molecule), which canreduce and/or inhibit a TLR8-induced cellular activity disclosed herein.In some embodiments, the TLR8 modulator is a TLR8 modulating chemicalcompound (e.g., small molecule), which can cause and/or enhance aTLR8-induced cellular activity disclosed herein. A TLR8 modulating(e.g., small molecule) may be a ligand that directly binds to TLR8.Alternatively, a compound may combine with TLR8 indirectly by, forexample, (a) forming a complex with another molecule that directly bindsto TLR8, or (b) otherwise causing the modification of another compoundso that the other compound directly binds to TLR8.

In some embodiments, the compound suppresses and/or reduces TLR8induced-cytokine production. In some embodiments, the compoundsuppresses and/or reduces extent and/or levels of cytokine production ascompared to, for example, extent and/or levels of cytokine producedduring untreated conditions (e.g., media alone or buffer alone). In someembodiments, the compound causes and/or enhancesTLR8 induced-cytokineproduction. In some embodiments, the compound causes and/or enhancesextent and/or levels of cytokine production as compared to, for example,extent and/or levels of cytokine produced during untreated conditions(e.g., media alone or buffer alone).

Polynucleotides

In some embodiments, the TLR8 modulator is a TLR8 modulatingpolynucleotide (e.g., polynucleotide).

In some embodiments, the TLR8 modulating polynucleotide is a TLR8polynucleotide agonist. In some embodiments, the TLR8 polynucleotideagonist causes and/or enhances a TLR8-induced cellular activity. In someembodiments, the TLR8 polynucleotide agonist further comprises a motif,which causes and/or enhances a TLR9-induced cellular activity such as anunmethylated CG or unmethylated TCG. In some embodiments, the TLR8polynucleotide agonist further comprises a motif, which causes and/orenhances a TLR7-induced cellular activity. In some embodiments, the TLR8polynucleotide agonist further causes and/or enhances a TLR7 and TLR9induced cellular activity. In some embodiments, the TLR8 polynucleotideagonist does not cause and/or enhance a TLR7 and/or TLR9 inducedcellular activity. In some embodiments, the TLR8 polynucleotide agonistcauses and/or enhances TLR8 induced-cytokine production. In someembodiments, the TLR8 polynucleotide agonist causes and/or enhancesextent and/or levels of cytokine production as compared to, for example,extent and/or levels of cytokine produced during untreated conditions(e.g., media alone or buffer alone). In some embodiments, the TLR8polynucleotide agonist causes and/or enhances TLR8 induced-immuneresponse. In some embodiments, the TLR8 polynucleotide agonist causesand/or enhances extent and/or levels of immune response as compared to,for example, extent and/or levels of immune response produced duringuntreated conditions (e.g., media alone or buffer alone).

In some embodiments, the TLR8 polynucleotide antagonist comprises a TLR8IRS. In some embodiments, the TLR8 IRS reduces and/or inhibits aTLR8-induced cellular activity. In some embodiments, the TLR8 IRSfurther comprises a motif which reduces and/or inhibits a TLR7 and/orTLR9-induced cellular function. In some embodiments, the TLR8 IRS doesnot comprise (i.e., lacks) a motif which reduces and/or inhibits a TLR7and/or TLR9-induced cellular function. In some embodiments, the TLR8 IRSdoes not comprise an immunostimulatory sequences such as unmethylated CGor unmethylated TCG. In some embodiments, the TLR8 modulator is not(i.e., excludes) an antisense oligonucleotide and/or does not operate bya RNAi pathway. In some embodiments, the TLR8 modulator is an antisenseoligonucleotide. In some embodiments, the TLR8 modulator is not amicroRNA or siRNA. In some embodiments, the TLR8 modulator is a microRNAor siRNA.

In some embodiments, the TLR8 IRS suppresses and/or reduces TLR8induced-cytokine production. In some embodiments, the TLR8 IRSsuppresses and/or reduces extent and/or levels of cytokine production ascompared to, for example, extent and/or levels of cytokine producedduring untreated conditions (e.g., media alone or buffer alone). In someembodiments, the TLR8 IRS suppresses and/or reduces TLR8 induced-immuneresponse. In some embodiments, the TLR8 IRS suppresses and/or reducesextent and/or levels of TLR8 induced-immune response as compared to, forexample, extent and/or levels of immune response produced duringuntreated conditions (e.g., media alone or buffer alone).

In some embodiments, a TLR8 modulating polynucleotide comprises an IRS,as described herein, which inhibits and/or suppresses a measurableimmune response as measured in vitro, in vivo, and/or ex vivo. In someembodiments, the TLR8 immune response is an innate TLR8 immune response.In some embodiments, the immune response is an adaptive TLR8 immuneresponse.

General Techniques

The practice of the present application employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, chemistry,biochemistry and immunology, which are within the skill of the art. Suchtechniques are explained fully in the literature, such as, MolecularCloning: A Laboratory Manual, second edition (Sambrook et al., 1989);Oligonucleotide Synthesis (Gait, ed., 1984); Animal Cell Culture(Freshney, ed., 1987); Handbook of Experimental Immunology (Weir &Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (Miller &Calos, eds., 1987); Current Protocols in Molecular Biology (Ausubel etal., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al.,eds., 1994); Current Protocols in Immunology (Coligan et al., eds.,1991); The Immunoassay Handbook (Wild, ed., Stockton Press NY, 1994);Bioconjugate Techniques (Hermanson, ed., Academic Press, 1996); andMethods of Immunological Analysis (Masseyeff, Albert, and Staines, eds.,Weinheim: VCH Verlags gesellschaft mbH, 1993).

EXAMPLES

Abbreviations: BAC (bacterial artificial chromosome); BM (bone marrow);CT (threshold cycle); CTRL (control); FACS (fluorescence activated cellsorter); hTLR8Tg (human Toll-like receptor 8 transgenic); IRS(immunoregulatory sequence); KO (knock out); MDC (myeloid dendriticcells); PBMC (peripheral blood mononuclear cells); PDC (plasmacytoiddendritic cells); PN (polynucleotides); TLR (Toll-like receptor); and WT(wild type).

Example 1 TLR8 Expression

TLR8 expression was analyzed in human cellular subsets (FIG. 1).Plasmacytoid dendritic cells (PDC), monocytes, myeloid dendritic cells(MDC), CD4+ T-cells, CD8+ T-cells, and neutrophils were purified bymeans of magnetic beads (Miltenyi Biotech) according to manufacturer'sinstructions. RNA was purified with a micro RNA KIT (Qiagen) accordingto manufacturer's instructions. cDNA from RNA was generated withSuperScript First-Strand Synthesis System (Invitrogen). Threshold cycle(CT) values for each gene were normalized to the housekeeping geneUbiquitin using the formula: relativeCT=1.8^((Avg CT Ubi-CT Gene))*100,000 where Avg CT Ubi is the mean CT oftriplicate housekeeping gene runs, Avg CT Gene is the mean CT ofduplicate runs of the gene of interest, and 100,000 is arbitrarilychosen as a factor to bring all values above one. Neutrophils showed thehighest level of expression. TLR8 was also expressed in monocytes, mDCsand CD4+ T-cells (FIG. 1).

Example 2 Activity of TLR7 and TLR8 RNA Polynucleotides (PN) in Humanand Mouse Cells

PN-based TLR8 ligand stabilized immunomodulatory RNA(5′-M2UGCUGCUUGUG-/glycerol/-GUGUUCGUCGUM2-5′ (M2=C6-linker); SEQ IDNO:3) and PN-based TLR7 ligand stabilized immunomodulatory RNA(5′-URCURCUUCUR-/glycerol/-RUCUUCRUCRU-5′ (R=7-deazaguanosine); SEQ IDNO:2) were previously identified (Lan et al., PNAS 104:13750-13755,2007). The effect of these TLR7- and TLR8-stimulating RNA PN wasevaluated by measuring production of IL-6 and TNF-α in human peripheralblood mononuclear cells (PBMCs) and human monocytes (FIG. 2). 5×10⁵PBMCs or 2×10⁵ monocytes from healthy human donors were stimulated with20 μg/mL, 100 μg/mL, or 200 μg/mL of either TLR8 agonist or TLR7 agonistas indicated in FIG. 2. Twenty-four hours later supernatants wereassessed for presence of inflammatory cytokines IL-6 and TNF-α bystandard ELISA procedure. TLR7 and TLR8 RNA PN stimulated production ofIL-6 and TNF-α in PBMCs and, to a lesser extent, in monocytes (FIG. 2).

The effect of TLR7 and TLR8-stimulating RNA PN were also evaluated inhuman pDCs by measuring production of IL-6, TNF-α, and IFN-α. As shownin FIG. 1, human pDCs were negative for the expression of TLR8. 1×10⁵pDCs from healthy human donors were stimulated with 100 μg/mL of eitherTLR8 agonist or TLR7 agonist (Lan et al., PNAS 104:13750-13755, 2007) ormedium alone as indicated in FIG. 3. Twenty-four hours latersupernatants were assessed for presence of inflammatory cytokines IL-6,TNF-α and IFN-α by standard ELISA procedure. The TLR8 agonist did notstimulate the pDCs proving its specificity for TLR8 (FIG. 3)

The effect of TLR7 and TLR8-stimulating RNA PNs was further evaluated inmouse cells by measuring IL-12 and TNF-α production (FIG. 4). Mousesplenocytes and PBMCs were prepared from 129 wt mice and TLR7KO mice,and 5×10⁵ cells were stimulated with 100 μg/ml of either TLR8 or TLR7agonist as indicated in FIG. 4. IL-12 and TNF-α were measured by ELISAusing standard techniques. The TLR8 agonist did not stimulate mousesplenocytes and PBMCs (FIG. 4).

Example 3 Generation of Human TLR8 (TLR8) Transgenic Mice

Transgenic TLR8 mice were generated using BAC/ES technologies(Sparwasser et al., Immunology 121:308-313, 2007). Briefly, the humanBAC carrying TLR8 gene was obtained from the RPCIB-753 BAC library. Thehuman BAC contains both human TLR7 and TLR8 genes in a cluster. The BACwas modified to knock out TLR7 gene to retain the chromosomal region atthe 5′UTR of mouse TLR8 that likely contains the promoter. Human TLR7gene was knocked out by inserting a neomycin cassette(FRT-PGK-gb2-neo-FRT). The FRT-PGK-gb2-neo-FRT template encoded theneomycin/kanamycin resistance gene, which combined a prokaryoticpromoter (gb2) for expression of kanamycin resistance in E. coli with aeukaryotic promoter (murine phosphoglucokinase gene (PGK)) forexpression of neomycin resistance in mammalian cells. A syntheticpolyadenylation signal inhibited the kanamycin/neomycin expression. Thecassette was flanked by FRT sites for later excision by Flp-recombinase.The appropriate modification was introduced into the BAC using bacterialrecombination procedures. After purification of the modified BAC, itsquality was checked using pulse-field gel electrophoresis and Southernblot analysis. Important regions of the gene of interest (such as exonsor proximal promoter), as well as the introduced modification wereconfirmed by sequencing. The sequence of constructBAC_RP11-1137P1_TLR7-KO: is provided as SEQ ID NO:1.

The modified BAC construct, BAC_RP11-1137P1_TLR7-KO (FIG. 5), wastransfected into an ES cell line, C57BL/6NTac ES cell line. TheC57BL/6NTac ES cell line was grown on a mitotically inactivated feederlayer comprised of mouse embryonic fibroblasts (MEF) in DMEM HighGlucose medium containing 20% FBS (PAN) and 1200 U/mL LeukemiaInhibitory Factor (Millipore ESG 1107). For manipulation 1.5×10⁵ EScells were plated on 3.5 cm dishes and transfected one day later withpurified BAC_RP11-1137P1_TLR7-KO DNA and Lipofectamine™ 2000 Reagentfrom Invitrogen (Catalog No. 11668-027) according to manufacturer'sprotocol. From day 2 onwards the medium was replaced daily with mediumcontaining 200 μg/mL G418 (Geneticin; Invitrogen; Catalog No.10131-019). Seven days later single clones were isolated, expanded andanalyzed by Southern Blot.

The ES clones generated were validated for presence of the TLR8 geneintegrated on the genome by using southern blot techniques. Five ESclones were chosen: Clone 8 and clone 6 with about 1-2 copies of TLR8integrated in the genome, Clone 12 with 2-4 copies of TLR8 integrated inthe genome, clone 16 with about 5 copies of TLR8 integrated in thegenome and Clone 23 with about 15 copies of TLR8 integrated in thegenome, respectively.

ES cells were injected into the blastocoel of 3.5 day old mouseblastocysts from BALB/c females. Subsequently, the injected embryos weretransferred to the uterine horns of appropriately timed pseudopregnantrecipient BALB/c females. Embryos gestated for about 18 days and theresulting pups were chimeras, whose tissues have developed from both theES cells carrying TLR8 gene and the recipient blastocyst cells of BALB/cbackground. This mix of starter cells was visible in the mouse's coat,which exhibited patches of coat color from the host embryo and patchesfrom the injected ES clone. Chimeras with 50-75% ES contribution (basedon fur color) were then put to breed with C57BL/6 animals to passgermline transmission.

Example 4 Analysis of TLR8 Chimeric Mice

The clones described above were evaluated in more detail. Chimeric ratiowas about 30% TLR8 transgene and 70% wild-type. Chimeric mice of fourout of five clones (clones 6, 12, 16 and 23) died before germlinetransmission. Only chimeric mice of clone 8 were able to breed and passgermline transmission. Clones 6, 12, 16, and 23 resulted in chimeralethality.

To determine TLR8 expression 200 μl of blood was harvested from mice andRNA and cDNA were prepared according to standard procedures. TAQMANassay was used to evaluate TLR8 gene expression. TLR8 expressioncorrelated with survival time (FIG. 6A-B). The highest expressing clones(6, 12, and 23) caused lethality of the chimeric mice. Only Clone 8 withabout 1-2 copies of TLR8 integrated in the genome survived (FIG. 6A-B)and passed germline transmission (TLR8TGCL8 mice). Additionally,increased inflammatory cytokine levels were detected in the serum ofTLR8Tg mice (n=15) as compared to control WT C57BL/6 mice (FIG. 6C).

Example 5 Gross and Histopathological Analysis of TLR8 Chimeras withHigh TLR8 Expression

To further evaluate the phenotype of the chimeras with high expressionof TLR8, biopsy specimens from chimeric mice of clone 12, 23, and 6 wereharvested from mice that showed evident sign of distress and needed tobe euthanized. Organs were fixed in formalin and embedded in paraffin.Organs from wild type C57BL/6 (CTRL B6) mice were also harvested ascontrols. Sections were stained with hematoxylin-eosin. Blindedevaluation of the liver, kidney, intestine, lung, brain, heart andpancreas was conducted by a pathologist during the course of the study.Inflammation was scored 1 to 4 as follow: 1=Minimal; 2=Mild; 3=Moderate;4=Marked. Statistical significance among groups was calculated with aMann-Whitney U-test with P values comparing chimeric mice to CTRL B6animals. P values were considered statistically significant at p≦0.05.

As shown in Table 5-1, histopathology of the TLR8 chimera's organsrevealed multi-organ inflammation with massive autoimmune pancreatitis.Inflammation was found in liver, kidney, and pancreas of human TLR8expressing mice. Pancreas histological data revealed abundantlymphocytes and macrophages/neutrophils infiltration, while acinar cellswere intact. Kidney histological data revealed interstitialinflammation, glomerular changes with segmental hypercellularity, andpyelitis renal pelvis.

TABLE 5-1 Histopathology of the TLR8 Chimeric Mice Organs Kidney LiverPancreas Glomerulo- Kidney Cholangio- ID Name Pancreatitis nephritisPyelitis hepatitis CL#12 1-1 3 2 0 1 CL#12 2-2 3 3 0 3 CL#23 138800 4 33 2 CL#23 3-1 4 — — 3 CL#23 4-1 4 3 2 4 CL#23 T4720 4 3 0 2 CL#23 1368334 3 3 0 CL#23 138782 4 3 2 1 CL#23 138785 4 3 3 2 CL#23 3-2 4 3 3 4CL#23 5-4 4 3 3 3 CL#23 T4715 4 3 3 1 Average 3.7 2.4 1.8 2.0 SEM 0.20.1 0.4 0.3 P value <0.001 <0.001 N.S. <0.001 CTRL B6 #1 0 0 0 0 CTRL B6#2 0 0 0 0 CTRL B6 #3 0 2 3 0 CTRL B6 #4 0 2 0 0 Average 0 1.0 0.6 0 SEM0 0.3 0.4 0

Example 6 Pathological Characterization of Chimeric Mice with High TLR8Expression

To further analyze the phenotype observed in chimeric mice with highexpression of TLR8, pancreatic cytokine production was determined (FIG.7). Pancreases from TLR8 chimeric mice of Clone 12, 6, or 23 (totalnumber of mice=6) or CTRL WT C57BL/6 mice (total number of mice=6) wereharvested and RNA was extracted with fibrous tissue RNA extraction kit(Qiagen) according to manufacturer's instructions. cDNA from RNA wasgenerated with SuperScript First-Strand Synthesis System (Invitrogen).Relative CT values for each gene were normalized to the housekeepinggene Ubiquitin using the formula: Geneexpression=1.8^((Avg CT Ubi-CT Gene))*100,000 where Avg CT Ubi is themean CT of triplicate housekeeping gene runs, Avg CT Gene is the mean CTof duplicate runs of the gene of interest, and 100,000 is arbitrarilychosen as a factor to bring all values above one. Results show a strongup-regulation of a number of inflammatory cytokines such as IFN-gamma,TNF-α, IL12, IP-10, IL-1α, and IL-1β in the chimeric mice compared tothe C57BL/6 control mice. (FIG. 7A-F).

Aberrant recognition of self RNA and DNA has been implicated in thedevelopment of pathogenic autoantibodies in human lupus patients and inmouse models of this disease. The presence of nucleic acid-specificautoantibodies in the serum of the TLR8 chimeric mice was assessed usingcommercially available kits from Alpha Diagnostic. Sera was collectedfrom TLR8 chimeric mice of clone 12 when the mice became moribund.Significantly increased levels of anti-ANA Ig, anti-dsDNA Ig andanti-RNP Ig were detected in the serum from the TLR8 chimeric mice (FIG.7G-I). Thus, over-expression of hTLR8 is associated with development ofanti-self nucleic acid antibodies.

In addition, myeloid dendritic cells (MDCs) were analyzed in chimericTLR8TGCL23 and wild type B57BL/WT mice (FIG. 8A-D). Chimeric andwild-type mice were sacrificed and spleens were harvested. Flowcytometric analyses were performed using fluorochrome-conjugatedmonoclonal antibodies to mouse CD11c, and costimulatory molecules CD80,GITRL, OX40L and CD40 (BD bioscience). These data show that MDCs werehighly activated inTLR8TGCL23 chimeric mice (FIG. 8).

Example 7 Analysis of Mice Receiving Bone Marrow from TLR8 Chimeras

To examine whether epithelial cells or leukocytes are responsible forthe phenotype observed in chimeric mice with high levels of TLR8expression, bone marrow (BM) from TLR8 chimeras of Clone 6, 12 and 23was transferred to recipient C57/BL6 (SJL) mice and animals weremonitored for BM uptake and pathology. Recipient mice (C57/BL6 (SJL))were irradiated with 900 rad using a cobalt irradiator. Four hourslater, 2×10⁶ BM cells extracted from the femurs of TLR8 chimeras weretransferred intravenously. Reconstitution of the BM of the recipientmice with the BM of TLR8 chimeras was verified using flow cytometry onblood samples. 25 mice were obtained of which the BM was reconstitutedwith 80% of TLR8 BM.

Mice that were transplanted with TLR8 transgenic mice bone marrow cells(TLR8TG>C57BL/6SJL mice) were monitored daily. Mice died very quicklywithin 30-60 days after introduction of the TLR8 BM or were euthanizedwhen moribund (FIG. 9). Wild-type mice reconstituted with bone marrowfrom huTLR8 chimeras developed a spontaneous inflammatory diseaseremarkably similar to that in the original ES chimeras. In contrast,wild type mice reconstituted with WT BM (C57BL/6>C57BL/6SJL mice) didnot show any signs of illness. These data suggest that high level TLR8expression in the hematopoietic compartment is sufficient to cause thephenotype observe in chimeric TLR8 mice.

TABLE 7-1 Histopathology of the TLR8TG> C57BL/6SJL Bone MarrowTransplanted Mice* Kidney Kidney Salivary Small Clone BM Pancreas D1 D2Liver Gland Intestine CL 23 4 1 0 1.5 3 NT CL 23 3.5 0 2 3 NT NT CL233.5 0 1 2.5 3.5 NT CL 6 NT 0 0 1.5 2 NT CL 6 4 0 0 2 2.5 NT CL 6 4 0 03.5 2 NT CL 12 4 0 0 1.5 3.5 0 CL 12 4 0 0 2 2.5 2.5 CL 12 4 0 0 1.5 3.52.5 CL 12 4 0 1 2.5 4 1 CL 12 4 0 0 2.5 3.5 0 CL 12 4 0 1 1.5 3.5 1 CL12 4 0 0 2 1.5 1 CL 12 4 0 0 2.5 3 0 CL 12 4 0 0 1.5 3 1 CL 12 4 0 0 1.52 1 CL 12 4 1 1 2 4 2.5 CL 12 4 0 0 2 1.5 3 CL 12 4 0 0 3 1.5 0 CL 12 40 NT 1.5 3.5 1 CL 12 4 0 0 2.5 3.5 3.5 CL 12 4 0 IS 1.5 2 2 CL 12 4 0 NT2 3.5 2 CL 12 4 0 0 2.5 2.5 2.5 CL 12 3.5 0 0 0 0.5 1 Average 3.9 0.10.3 2.1 2.7 1.4 SEM 0.03 0.06 0.11 0.14 0.19 0.25 P value <0.001 <0.0010.61 <0.001 <0.001 0.23 B6 CTRL 0 0 0 1 0 1 B6 CTRL 0 1 0 1 0 0 B6 CTRL0 1 0 1 0 1 B6 CTRL 0 1 0.5 1 0 1 Average 0.0 0.8 0.1 1.0 0.0 0.8 SEM0.00 0.25 0.13 0.00 0.00 0.25 *Gross Pathology: pancreas pancreatitis,kidney D1 glomerulonephritis, kidney D2 pyelitis, livercholangiohepatitis, salivary gland inflammation, and small intestineinflammation.

To evaluate the histopathology of TLR8TG>C57BL/6SJL mice, biopsyspecimens were harvested from mice that showed evident signs of distressand needed to be euthanized. Organs were fixed in formalin and embeddedin paraffin. Organs from C57BL/6SJL (CTRL B6) mice were also harvestedas controls. Sections were stained with hematoxylin-eosin. Blindedevaluation of the liver, kidney, intestine, lung, brain, heart andpancreas were conducted by a pathologist. Inflammation was scored 1 to 4as follow: 1=Minimal; 2=Mild; 3=Moderate; 4=Marked. Statisticalsignificance among groups was calculated with a Mann-Whitney U-test. Pvalues compare chimeric mice to CTRL B6 animals. P values are consideredstatistically significant at p≦0.05. As shown in Table 7-1histopathology of the TLR8TG>C57BL/6SJL bone marrow transplanted micerevealed multi-organ inflammation with massive autoimmune pancreatitis.

Flow cytometric analyses of T cells of TLR8TGCL12>C57BL/6SJL andwild-type C57BL/6SJL mice were performed using fluorochrome-conjugatedmonoclonal antibodies to mouse CD4, CD8, CD44, CD62L (BD bioscience)(FIG. 10A-B). 30-40 days after bone marrow transplantation,TLR8TG>C57BL/6SJL mice (n=19) were sacrificed and blood and spleens wereharvested. C57BL/6SJL WT mice (n=9) were used as controls. Dot blotanalysis (FIG. 10) and cumulative data (FIG. 11) show that naive cellswere CD44 low and CD62L high, effector cells were CD44 high and CD62Lhigh, and effector memory cells were CD44 high and CD62L negative. Pvalues were calculated with a Mann-Whitney U-test and were consideredstatistically significant at p≦0.05. The presence of effector andeffector memory cells shows that T cells were highly activated inTLR8TG>C57BL/6SJL mice.

In addition, flow cytometric analyses of MDC of TLR8TG>C57BL/6SJL andwild-type C57BL/6SJL mice were performed using fluorochrome-conjugatedmonoclonal antibodies to mouse CD11c, and costimulatory molecules CD80,GITRL, OX40L, PDL-1, and MHC CLASS II (BD bioscience) (FIG. 12).TLR8TG>C57BL/6SJL mice were sacrificed and spleens were harvested.C57BL/6SJL wild-type mice were used as controls.

These data suggest that high level TLR8 expression in leukocytes and notepithelial cells is responsible for the phenotype observed in chimericTLR8 mice. In these mice, the bone-marrow originated from theTLR8-transgenic animal while the hosts are wild-type. Therefore, thismeans that the entire hematopoietic compartment carries the transgenebut the tissues (and thus all epithelial cell) are wild-type.

Example 8 Transgenically Expressed TLR8 is Functional

PN-based TLR8 ligand stabilized immunomodulatory RNA(5′-M2UGCUGCUUGUG-/glycerol/-GUGUUCGUCGUM2-5′ (M2=C6-linker); SEQ IDNO:3) and PN-based TLR7 ligand stabilized immunomodulatory RNA(5′-URCURCUUCUR-/glycerol/-RUCUUCRUCRU-5′ (R=7-deazaguanosine); SEQ IDNO:2) were previously identified (Lan et al., PNAS 104:13750-13755,2007). To confirm that human TLR8 expressed from the transgene inTLR8TGCL8 mice is functional and sensitive to stimulation, the effect ofTLR8-stimulating RNA PN (SEQ ID NO:3) on cytokine production wasassessed. 5×10⁵ blood cells from TLR8TGCL8 transgenic mice or C57BL/6 WTanimals were stimulated with TLR8 agonist at different concentrations asshown in FIG. 13. 24 hours later supernatants were harvested and assayedfor mouse IL-12, TNF-α and IL-6 using standard ELISA procedures. Inaddition, 5×10⁵ bone marrow cells from TLR8TGCL8 transgenic mice orC57BL/6 WT animals were stimulated with TLR8 agonist (SEQ ID NO:3) atdifferent concentrations as shown in FIG. 14. Mouse TNF-α and IL-6production was assayed in supernatants harvested after 24 hours. Onlycells from transgenic animals responded to the TLR8 agonistdemonstrating that human TLR8 is functional in these mice (FIGS. 13 and14).

Example 9 TLR8 Mediated Diseases

The role of TLR8 in disease and/or disease susceptibility was evaluatedby using the TLR8 over-expressing mice and/or bone marrow from the TLR8over-expressing mice as described below.

Spontaneous Arthriti.

hTLR8Tg chimeric mice from clone 12 (n=6) and C57BL/6 CTLR animals (n=6)were euthanized 90 days after the birth. Paws and joints were fixed,sectioned and stained with toluene blue. Representative sections ofjoints from WT mice (FIG. 15A) and hTLR8Tg mice (FIG. 15B) wereexamined. All of the hTLR8Tg mice showed signs of swelling in fore limbsand hind limbs. The histological changes, degree of inflammation andcartilage damage were evaluated by a pathologist in a blinded fashionand scored 1 to 5 as followed: 1=minimal, 2=mild, 3=moderate, 4=marked,5=severe. Two paws and two ankles were evaluated for each animal and thescores were summed to obtain the histological disease score (FIG. 15C).

Rheumatoid Arthritis

To investigate whether TLR8 had a role in rheumatoid arthritis,wild-type (C57BL/6) and TLR8 transgenic mice (TLR8TGCL8) were immunizedper a published immunization schedule and protocol (Campbell, Eur JImmunol 30:1568-1575, 2000). On day 0 of collagen immunization, collagen(Chicken Type II Collagen from Chondrex; 2 mg/mL) was emulsified withComplete Freund's Adjuvant (CFA from Chondrex; 5 mg/mL concentration ofMycobacterium tuberculosis H37Ra) as follows:

(i) one volume of CFA was mixed with an equal amount of the collagensolution;

(ii) mixing was continued until a stable, stiff emulsion resulted;

(iii) to ascertain the desired stability of the emulsion, 1 drop ofemulsion was added into a water-filled beaker (the emulsion wasconsidered stable if it remained in the water as a solid); and

(iv) 100 μl was injected subcutaneously at the base of the tail.

A second injection was performed at day 21. Animals were assessed forredness and swelling of the 4 limbs and the cumulative score of eachmouse was the sum of the score obtained for each limb. The ClinicalScore Guidelines were as follows: 0-Normal; 1-Mild, but definite rednessand swelling of the ankle or wrist, or apparent redness and swellinglimited to individual digits, regardless of the number of affecteddigits; 2-Moderate redness and swelling of ankle of wrist; 3-Severeredness and swelling of the entire paw including digits; and 4-Maximallyinflamed limb with involvement of multiple joints and a clinical scoreallocated.

Animals were sacrificed 80 days after CIA induction. The two frontjoints of each animal were used to prepare RNA to measure expression ofvarious genes using TAQMAN assays (46 joints per group). The data shownare cumulative of two independent experiments. The TLR8 transgenic micehad a higher cumulative clinical score and a significant increase in theincidence of symptoms with a clinical score 4 or greater over time thanwild-type mice (FIG. 16A-B). Based on this data, expression of TLR8 wasconfirmed to play a role in the development of rheumatoid arthritis.FIG. 16C-E shows significantly elevated expression of hTLR8, F4/80 andTNF in the joints of TLR8 transgenic mice (TLR8TGCL8) as compared towild-type mice (C57BL/6). In addition, the level of expression of hTLR8was found to correlate with the clinical score (FIG. 16F). Moreover,expression of F4/80 and TNF correlated with levels of hTLR8 expression(FIG. 16G-H). Likewise, expression of various pro-inflammatory cytokines(e.g., IL-1B, IL1A, IL-6, IL-18, IP-10, MMP-9) was found to correlatewith hTLR8 levels (data not shown).

Diabetes

To investigate whether TLR8 had a role in diabetes, C57BL/6-SJL WT Micewere transplanted with WT C57BL/6 bone marrow cells or with TLR8TGCL12bone marrow cells as described Example 7. Mice were monitored daily.Blood was collected from moribund mice, and glucose levels in the bloodwere assayed. Blood glucose levels in mice reconstituted with TLR8 BMwere significantly elevated as shown in FIG. 17. 62% of the mice showeda level equal or exceeding 200 mg/dL and were therefore considereddiabetic (See Lee et al., Nutrition Research 26:474-479, 2006; Serranoet al., Proc. Intl. Soc. Mag. Reson. Med. 15 2463, 2007; and Tanquilutet al., J. of Med. Plants Research 3: 1066-1071, 2009, which areincorporated by reference in their entirety).

Blood Disorders

Cellular subsets were isolated from spleens of B6.SJL mice transplantedwith bone marrow from TLR8TgCL12 mice. Expression of hTLR8 was evaluatedby TAQMAN. Cumulative data from at least three independent experimentsis shown (n=5-10, mean±SEM). Human TLR8 is expressed in monocytes,neutrophils and dendritic cells of the hTLR8Tg mice (FIG. 18A). Theexpression pattern huTLR8 in leukocyte subsets from these mice is verysimilar to its expression in human leukocytes.

Approximately 5×10⁵ PBMC from TLR8Tg chimeras or C57BL/6 WT animals werestimulated with a TLR8 agonist and 24 hr later supernatants were harvestand assayed for cytokine levels by ELISA (n=4 mice, mean±SEM). TheRNA-based TLR8 agonist ORN-8L (300 μg) induced secretion of IL-12 andTNF by PBMC from hTLR8Tg mice (FIG. 18B-C).

Mice from TLR8Tg Clone 8 line were injected intravenously with a TLR8agonist. Serum was collected 6 hr later and assayed for cytokine levels(n=3 mice, mean±SEM). The RNA-based TLR8 agonist ORN8-L (300 μg) or thesmall molecule-based TLR8 agonist CL075 (5 μg) induced secretion ofIL-12 in vivo in hTLR8Tg mice. Thus human TLR8 is functional in thesemice

The number of various white blood cells was assessed in peripheral bloodand in spleens of TLR8Tg chimeras or age-matched C57BL/6 controls (FIG.19A-B). TLR8Tg mice develop lymphopenia (decrease in number ofcirculating lymphocytes), granulocytosis (increase in number ofgranulocytes, which are primarily neutrophils) and monocytosis (increasein number of monocytes). As shown in FIG. 19C there is a significantdecrease in circulating lymphocytes paralleled by an increase ingranulocytes and monocytes in the blood of the TLR8 expressing mice ascompared to WT controls.

Cells from spleens from TLR8Tg clone 12 chimeric mice and from WT micewere stimulated in vitro for 2 hr with PMA (5 ng/ml) and ionomycin (500ng/ml). The percentage of CD4 and CD8 T cells producing TNF andIFN-gamma was increased in TLR8Tg chimeric mice as compared to CD4 andCD8 T cells from wild type control mice (FIG. 20A-C).

Example 10 Screening for and/or Identifying TLR8 Antagonists andAgonists

TLR8 antagonists and agonist can be screened for and/or identified usingthe transgenic mice expressing human TLR8 described above.

In Vitro Assays

Human primary monocytes and splenocytes from TLR8TGCL8 mice are used toscreen for human TLR8 antagonists and agonists. Cells are stimulatedwith TLR8-specific ligands and the activation or inhibition ofTLR8-related immune responses are measured by standard in vitro assaysdescribed herein (e.g., cytokine production, cell proliferation, markergene expression, and/or cell surface markers).

In Vivo Assays

Clone 8 TLR8 transgenic mice either with germline transmission or TLR8chimeras or without germline transmission are used to screen for TLR8antagonists and agonists. Furthermore, new chimeras can be generatedusing TLR8 transgenic mice with high copy number TLR8 ES expressingclones. Mice are injected with either TLR8 agonist or antagonistcandidates. TLR8 agonists activate, while TLR8 antagonists inhibit theTLR8-dependent signal transduction pathway and TLR8-associated immuneresponses. Mice are injected with TLR8 agonist known in the art prior toinjection of TLR8 antagonist candidate. Alternatively, TLR8 antagonistcandidates can be assessed in spontaneous disease mouse models in theabsence of administration of a TLR8 agonist

Autoimmune mouse models employing the TLR8 transgenic mice of thepresent disclosure include, for example, models for rheumatoidarthritis, diabetes, pancreatitis, glomerulonephritis, pyelitis,cholangiohepatitis, and reproductive disorders.

1-6. (canceled)
 7. A hematopoietic cell obtained from a transgenic mousewhose genome comprises a nucleotide sequence encoding human Toll-likereceptor 8 (TLR8), wherein said human TLR8 is expressed in the cell, andwherein at least one inflammatory cytokine selected from the groupconsisting of tumor necrosis factor-alpha (TNF-alpha), interleukin-6(IL-6), interleukin-17 (IL-17), interleukin-12 p40 (IL-12B), chemokinecc motif ligand 2 (CCL-2) and interferon-gamma-inducible protein 10(IP10) is present in sera from the transgenic mouse at an elevated levelas compared to a control, non-transgenic mouse. 8-12. (canceled)
 13. Amethod of screening candidate agents, the method comprising: contactingthe cell of claim 7 with a candidate agent; and determining the effectof said candidate agent on a TLR8-mediated response of said cell. 14.The method of claim 13, wherein the effect comprises inhibition of saidTLR8-mediated response.
 15. The method of claim 13, wherein the effectcomprises stimulation of said TLR8-mediated response.
 16. The method ofclaim 13, wherein the effect is evidenced by a change in TLR8-mediatedcytokine production, cell proliferation, and/or cell surface markerexpression.
 17. The method of claim 16, wherein the effect is evidencedby a change in TLR8-mediated production of one or more cytokines of thegroup consisting of tumor necrosis factor-alpha (TNF-alpha),interferon-alpha (IFN-alpha), interferon-beta (IFN-beta),interferon-gamma (IFN-gamma), interleukin-1alpha (IL-1alpha),interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-12(IL-12), interferon-gamma-inducible protein 10 (IP10), and macrophageinflammatory protein-1alpha (MIP-1alpha).
 18. The method of claim 16,wherein the effect is evidenced by a change in TLR8-mediated productionof one or more cytokines of the group consisting of tumor necrosisfactor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-12(IL-12).
 19. The method of claim 16, wherein the effect is evidenced bya change in TLR8-mediated cell proliferation.
 20. The method of claim16, wherein the effect is evidenced by a change in TLR8-mediatedexpression of one or more cell surface markers selected from the groupconsisting of CD40, CD80, CD86, glucocorticoid-induced tumor necrosisfactor receptor family-related protein ligand (GITRL), OX40 ligand(OX40L), and programmed death ligand-1 (PDL-1).
 21. The method of claim13, wherein the candidate agent is an antibody.
 22. The method of claim13, wherein the candidate agent is a small molecule.
 23. The method ofclaim 13, wherein the candidate agent is a polynucleotide.
 24. The cellof claim 7, wherein said transgenic mouse is a chimeric transgenic mousein which both human TLR8 and mouse TLR8 are expressed.
 25. The cell ofclaim 7, wherein said nucleotide sequence encoding human TLR8 is presentat a copy number of from 1 to
 5. 26. The cell of claim 7, wherein saidnucleotide sequence encoding human TLR8 is present at a copy number offrom 1 or
 2. 27. The cell of claim 7, wherein the hematopoietic cell isa population of cells comprising bone marrow.
 28. The cell of claim 7,wherein the hematopoietic cell is a population of cells comprisingsplenocytes.
 29. The cell of claim 7, wherein the hematopoietic cell isa population of cells comprising one or more of monocytes, myeloiddendritic cells and neutrophils.